Immobilised biological entities

ABSTRACT

There is described inter alia a device having a surface comprising a layered coating wherein the outer coating layer comprises a plurality of cationic hyperbranched polymer molecules characterized by having (i) a core moiety of molecular weight 14-1,000 Da (ii) a total molecular weight of 1,500 to 1,000,000 Da (iii) a ratio of total molecular weight to core moiety molecular weight of at least 80:1 and (iv) functional end groups, whereby one or more of said functional end groups have an anti-coagulant entity covalently attached thereto.

The present application is a continuation of U.S. Serial No. 15/204,128,filed Jul. 7, 2016, which is a division of U.S. Serial No. 14/790,205,filed Jul. 2, 2015, now U.S. Patent No. 9,408,950 issued Aug. 9, 2016,which is a division of U.S. Serial No. 13/416,880, filed Mar. 9, 2012,now U.S. Patent No. 9,101,696, issued Aug. 11, 2015, that claims thebenefit under 35 U.S.C. § 119 of U.S. Provisional Application Serial No.61/451,732, filed Mar. 11, 2011. The contents of both applications areherein incorporated by reference in their entireties.

This invention relates to immobilised biological entities to deviceshaving surface coatings comprising such entities, and processes andintermediates for their production. In particular, the invention relatesto immobilised anti-coagulant entities such as heparin and to devices,for example medical, analytical and separation devices having surfacecoatings comprising immobilised heparin.

BACKGROUND OF THE INVENTION

When a medical device is placed in the body, or in contact with bodyfluids, a number of different reactions are set into motion, some ofthem resulting in the coagulation of the blood in contact with thedevice surface. In order to counteract this serious adverse effect, thewell-known anti-coagulant compound heparin has for a long time beenadministered systemically to patients before the medical device isplaced in their body, or when it is in contact with their body fluids,in order to provide an antithrombotic effect.

Thrombin is one of several coagulation factors, all of which worktogether to result in the formation of thrombi at a surface in contactwith the blood. Antithrombin (also known as antithrombin III) (“AT” or“ATIII”) is the most prominent coagulation inhibitor. It neutralizes theaction of thrombin and other coagulation factors and thus restricts orlimits blood coagulation. Heparin dramatically enhances the rate atwhich antithrombin inhibits coagulation factors.

However, systemic treatment with high doses of heparin is oftenassociated with serious side-effects of which bleeding is thepredominant. Another rare, but serious complication of heparin therapyis the development of an immunological response called heparin inducedthrombocytopenia (HIT) that may lead to thrombosis (both venous andarterial). High dose systemic heparin treatment e.g. during surgery alsorequires frequent monitoring of the activated clotting time (used tomonitor and guide heparin therapy) and the corresponding doseadjustments as necessary.

Therefore solutions have been sought where the need for a systemicheparinisation of the patient would be unnecessary or can be limited. Itwas thought that this could be achieved through a surface modificationof the medical devices using the anti-coagulant properties of heparin.Thus a number of more or less successful technologies have beendeveloped where a layer of heparin is attached to the surface of themedical device seeking thereby to render the surface non-thrombogenic.For devices where long term bioactivity is required, the heparin layershould desirably be resistant to leaching and degradation.

Heparin is a polysaccharide carrying negatively charged sulfate andcarboxylic acid groups on the saccharide units. Ionic binding of heparinto polycationic surfaces has been attempted, but these surfacemodifications tended to suffer from lack of stability over timeresulting in lack of non-thrombogenic function, as the heparin leachedfrom the surface.

Thereafter different surface modifications have been prepared whereinthe heparin has been covalently bound to groups on the surface.

One of the most successful processes for rendering a medical devicenon-thrombogenic has been the covalent binding of a heparin fragment toa modified surface of the device. The general method and improvementsthereof are described in European patents: EP-B-0086186, EP-B-0086187,EP-B-0495820 and U.S. Pat. No. 6,461,665.

These patents describe the preparation of surface modified substrates byfirst, a selective cleavage of the heparin polysaccharide chain, e.g.using nitrous acid degradation, leading to the formation of terminalaldehyde groups. Secondly, the introduction of one or more surfacemodifying layers carrying primary amino groups on the surface of themedical device, and thereafter reacting the aldehyde groups on thepolysaccharide chain with the amino groups on the surface modifyinglayers followed by a reduction of the intermediate Schiff's bases toform stable secondary amine bonds.

Other methods of modifying surfaces are known. For example, US2005/0059068 relates to a substrate for use in microassays. An activatedpolyamine dendrimer is covalently bonded to the surface of the substratethrough a silane containing moiety. The dendrimer has branch pointswhich are tertiary amines and terminal residues which are NH₂, OH, COOHor SH groups. Molecules containing OH or NH₂ functional groups can bebound to the dendrimer via the terminal residues of the dendrimer. Sincethe substrate is for use in microassays, it is usually a slide, bead,well plate, membrane etc. and the moiety containing the OH or NH₂ groupis a nucleic acid, protein or peptide.

WO 03/057270 describes a device, for example a contact lens, with alubricious coating having high surface hydrophilicity. A number ofexamples of coating materials are given including glycosaminoglycans(e.g. heparin or chondroitin sulfate) and PAMAM dendrimers. PAMAMdendrimers are said to be among the preferred coatings. The documentexemplifies a contact lens having multiple layers of PAMAM dendrimer andpolyacrylamide-co-polyacrylic acid copolymer (PAAm-co-PAA). The coatingis formed by consecutively dipping the contact lens into solutions ofthe two coating materials, with the outer layer being PAAm-co-PAA.

US 2003/0135195 teaches a medical device such as a catheter with ahighly lubricious hydrophilic coating formed from a mixture of colloidalaliphatic polyurethane polymer, an aqueous dilution ofpoly(1-vinylpyrrolidone-co-2-dimethylaminoethylmethacrylate)-PVP anddendrimers. The document teaches that the coating may be applied to thedevice by dipping the device in a colloidal dispersion of the aliphaticpolyurethane polymer in a solution ofpoly(1-vinylpyrrolidone-co-2-dimethylaminoethylmethacrylate)-PVP and anactive agent (e.g. heparin) in a mixture of dendrimer, water,N-methyl-2-pyrrolidone and triethylamine. The document teaches thatheparin may be contained in the voids within the dendrimers. Thedocument also teaches that the loaded heparin will elute from thehydrophilic polymer matrix at a predetermined rate.

US 2009/0274737 teaches implants such as stents having a hydrophilicsurface with a wetting angle of ≤80°. There may be one, two or moreanti-coagulant ingredients permanently bound to the surface and examplesof anticoagulants include heparin and certain dendrimers, especiallysulphated dendrimers. The surface may be functionalised in order to bindthe anticoagulant and examples of functionalization are silanization andreaction with 1,1′-carbonyldiimidazole (CDI).

U.S. Pat. No. 4,944,767 relates to a polymeric material which is able toadsorb high quantities of heparin. The material is a block copolymer inwhich polyurethane chains are interconnected with polyamidoamine chains.

Our earlier application WO 2010/029189 relates to a medical devicehaving a coating with an anticoagulant molecule such as heparincovalently attached to the coating via a 1,2,3-triazole linkage. Thedocument describes the azide or alkyne functionalisation of a polyamine;the preparation of alkyne or azide functionalised heparin (both nativeand nitrous acid degraded heparin); and the reaction to link thederivatised heparin to the derivatised polymer via a 1,2,3-triazolelinker.

The product described in WO 2010/029189 has many advantages but we havesought to develop an improved material in which the bioavailability ofthe heparin or other attached anti-coagulant molecule is increased,which may have greater stability on aging and which can be manufacturedby a process which is robust and produces a product of high consistency.

Heparins have the ability to bind a wide variety of biomoleculesincluding enzymes, serine protease inhibitors (such as antithrombin),growth factors and extracellular matrix proteins, DNA modificationenzymes and hormone receptors. If used in chromatography, heparin is notonly an affinity ligand but also an ion exchanger with high chargedensity. Thus biomolecules can be specifically and reversibly adsorbedby heparins immobilized on an insoluble support. Immobilised heparinstherefore have a number of useful non-medical applications, particularlyfor analysis and separation.

SUMMARY OF THE INVENTION

According to the invention we provide, inter alia, a device having asurface comprising a layered coating wherein the outer coating layercomprises a plurality of cationic hyperbranched polymer moleculescharacterized by having (i) a core moiety of molecular weight 14-1,000Da (ii) a total molecular weight of 1,500 to 1,000,000 Da (iii) a ratioof total molecular weight to core moiety molecular weight of at least80:1 (e.g. at least 100:1) and (iv) functional end groups, whereby oneor more of said functional end groups have an anti-coagulant entitycovalently attached thereto.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation in 2-D of different types ofhyperbranched polymers in which A represents a polymer with branchingpoints (theoretically) in every monomeric unit; B represents a branchedpolymer with linear backbone and branched wedges, called dendrons,attached to it; C represents a polymer with branching units incorporatedinto linear segments; and D represents a dendrimer.

FIG. 2 illustrates in 2-D an exemplary PAMAM dendrimer having 3generations (in 3-D the structure would be approximately spherical).

FIG. 3 is a schematic illustration in 2-D of an exemplary secondgeneration dendrimer in which the core has three reactive functionalgroups, all of which are substituted; the first layer has six reactivefunctional groups, all of which are substituted; and the second layerhas twelve reactive functional groups. Such a dendrimer will adopt asubstantially spherical shape in 3-D.

FIG. 4 illustrates how a first functional group on a heparin moiety (orother anti-coagulant entity) may be reacted with a second functionalgroup which is an end group of the dendrimer or other hyperbranchedpolymer.

FIG. 5 shows how several dendrimers or other hyperbranched polymers maybe cross-linked to each other prior to functionalisation by heparin orother anti-coagulant entity.

FIG. 6 shows how several dendrimers or other hyperbranched polymerswhich have been functionalised by heparin or other anti-coagulant entitymay be cross-linked to each other.

FIG. 7 is a schematic representation of the components of the invention.It shows how the hyperbranched polymers, bearing anti-coagulantentities, in the outer coating layer interact (involving covalent bondsand/or ionic interactions) with the underlayer and other hyperbranchedpolymers in the outer coating layer.

FIG. 8 shows the percentage of platelets remaining in blood aftercontact with various non-thrombogenic coatings (see Example 6).

FIG. 9 shows an exemplary Toluidine blue staining of a PVC tube beforeand after coated with a heparin-containing coating according to theinvention (see Example 3.2 and Example 6.3). In FIG. 9; plate A: Before;plate B: After.

FIG. 10 shows the percentage of platelets remaining in blood aftercontact with various non-thrombogenic coatings (see Example 11).

DETAILED DESCRIPTION OF THE INVENTION

Anti-Coagulant Entities

An anti-coagulant entity is an entity capable of interacting withmammalian blood to prevent coagulation or thrombus formation.

Anti-coagulant entities are well known to those skilled in the art andmany of them are oligosaccharides or polysaccharides. Some of theentities are glycosaminoglycans including compounds containingglucosamine, galactosamine, and/or uronic acid. Among the most suitableglycosaminoglycans are “heparin moieties” and especially full lengthheparin (i.e. native heparin).

The term “heparin moiety” refers to a heparin molecule, a fragment ofthe heparin molecule, or a derivative or analogue of heparin. Heparinderivatives can be any functional or structural variation of heparin.Representative variations include alkali metal or alkaline earth metalsalts of heparin, such as sodium heparin (e.g. Hepsal or Pularin),potassium heparin (e.g. Clarin), lithium heparin, calcium heparin (e.g.Calciparine), magnesium heparin (e.g. Cutheparine), and low molecularweight heparin (prepared by e.g. oxidative depolymerization ordeaminative cleavage, e.g. Ardeparin sodium or Dalteparin). Otherexamples include heparan sulfate, heparinoids, heparin based compoundsand heparin having a hydrophobic counter-ion. Other desirableanti-coagulant entities include synthetic heparin compositions referredto as “fondaparinux” compositions (e.g. Arixtra from GlaxoSmithKline)involving antithrombin-mediated inhibition of factor Xa. Additionalderivatives of heparin include heparins and heparin moieties modified bymeans of e.g. mild nitrous acid degradation (U.S. Pat. No. 4,613,665) orperiodate oxidation (U.S. Pat. No. 6,653,457) and other modificationreactions known in the art where the bioactivity of the heparin moietyis preserved.

Heparin moieties also include such moieties bound to a linker or spaceras described below. De-sulphated heparin, or heparin functionalized viae.g. the carboxylic acid group of the uronic acid moiety, are lesssuitable than other forms of heparin because of their generally reducedanti-coagulant properties relative to other forms of heparin.Mono-functionalization or low functionalization degrees of carboxylicacid groups can be acceptable as long as heparin bioactivity ispreserved.

Suitably, each anti-coagulant entity is single point attached to ahyperbranched polymer molecule, particularly end point attached. Theattachment is via functional end groups on the hyperbranched polymermolecule as discussed below. When the anti-coagulant entity is an endpoint attached heparin moiety, it is suitably connected to thehyperbranched polymer molecule through its reducing end (sometimesreferred to as position C1 of the reducing terminal). The advantage ofend point attachment, especially reducing end point attachment, is thatthe biological activity of the anti-coagulant entity (for example theheparin moiety) is maximized due to enhanced availability of theantithrombin interaction sites as compared with attachment elsewhere inthe anti-coagulant entity (e.g. heparin moiety).

Where there is a multiplicity of anti-coagulant entities e.g. heparinmoieties it is possible for some or all of them to be of a differenttype; however generally they will all be of the same type.

Anti-coagulant entities are commonly anionic (as in the case of heparinmoieties).

Other anti-coagulant entities such as hirudin, coumadins (vitamin Kantagonists of the 4-hydroxycoumarin class like warfarin), anti-plateletdrugs (as clopidogrel and abciximab), argatroban, thrombomodulin oranti-coagulant proteins (like proteins C, S or antithrombin) may also beconsidered for use. Anti-coagulant entities may also include enzymessuch as apyrase. Such substances may be charged (e.g. anionic) oruncharged. The way these may be attached to the hyperbranched polymer sothat its bioactivity is preserved can be designed by someone skilled inthe art.

Hyperbranched Polymers

Examples of various types of hyperbranched polymers are shownschematically in FIG. 1, types A to D. A, in FIG. 1, represents apolymer with branching points (theoretically) in every monomeric unit; Brepresents a branched polymer with linear backbone and branched wedges,called dendrons, attached to it; C represents a polymer with branchingunits incorporated into linear segments; and D represents a dendrimer.These polymers are examples of hyperbranched polymers useful in thecontext of the present invention if the core segment is sufficientlysmall in relation to the overall size of the molecule.

The term “hyperbranched polymer molecule” is well understood in the artto refer to a molecule having a tree like branching structure emanatingfrom a core moiety typically in the centre. In the context of thepresent invention, the term also includes dendrimers, which are wellknown and are hyperbranched polymer molecules in which the degree ofbranching is 100% (occasionally referred to herein as “perfectlybranched” i.e. 100% of functional groups capable of branching arebranched) and which are therefore highly symmetrical about the core.Hyperbranched polymers consist of three basic architectural components,(i) the core, (ii) the interior and (iii) the functional end groups. Thecore is positioned at the centre of the molecule and to it branchedwedges, called dendrons, are attached. The dendrons may be perfectlybranched or less than perfectly branched.

The core of a hyperbranched polymer molecule is polyfunctional (eitherwith several of the same type or several of different types offunctionalities) and the number of functional groups it bears dictatesthe number of branches possible to be introduced in the molecule.Typically all functional groups of the core are utilized in branching.Similarly, the shape of a hyperbranched polymer molecule is determinedby the core shape, with substantially tetrahedral cores giving rise tosubstantially spherical hyperbranched polymer molecules and moreelongated cores giving rise to ovoid or rod-shaped hyperbranched polymermolecules.

According to the invention, the core moiety will be a relatively smallentity in relation to the overall size of the polymer, having amolecular weight of between 14 and 1,000 Da, more usually between 40 and300 Da and for example 50 to 130 Da.

Dendrimers are perfectly branched molecules in which the degree ofbranching is 100%, thus their structure is highly regular and therefore,for a given starting material, the only variable is the number of layersor generations in the dendrimer. The generations are conventionallynumbered outwards from the core. See, for example, Tables 2-4 below.FIG. 2 illustrates a third generation dendrimer and FIG. 3 illustrates asecond generation dendrimer. Because of their highly consistent andsymmetrical structure, the molecular weight distribution for dendrimersof a given generation is extremely narrow, which is highly advantageousas it leads to a very consistent product.

Other hyperbranched molecules also contain a high number of branches,however, and, for example the degree of branching will usually be atleast 30%, 40% or 50% for example at least 60%, 70%, 80% or 90%. Unlikedendrimers, the structure of such hyperbranched molecules will not becompletely regular but they may also adopt a generally globularstructure.

Typically the core is the moiety of a molecule which is not the same asthe repeating unit(s) of the polymer. However in one embodiment the coreis a moiety of the same type as the repeating unit (or one of therepeating units) of the polymer.

Hyperbranched polymer molecules are generally prepared either byemploying a divergent method, in which the layers are built up from acore, or a convergent method in which fragments are built up and thencondensed. Dendrimers are more usually prepared using the divergentmethod.

In the synthesis of dendrimers a high degree of control over theaddition reaction of every branching unit is essential and the resultingproducts show a polydispersity index (PDI) between 1.00 and 1.05. Thedendron size depends on the number of monomer layers and every addedlayer is represented by a generation (G). The interior consist ofbranching monomers that have ABx functionality where x≥2. Carefulpreparation of the branching unit makes it possible to control thereaction between A and B′ if B′ is the activated state of B. Largerdendrimers give rise to globular shaped, nanoscale sized, structureswith low intrinsic viscosity as a result.

Traditionally, dendrimers are synthesized by employing an iterativetechnique where ABx monomers are alternately added to the growingspecies followed by an activation/deprotection step. These protocolsdepend on efficient reactions that ensure full substitution of theterminal groups B′. Any deviation will give structural defects thataccumulate during dendrimer growth resulting in tedious or impossiblepurification procedures.

See Aldrichimica Acta (2004) 37(2) 1-52 “Dendrimers: building blocks fornanoscale synthesis”, herein incorporated in its entirety by reference,e.g. at pages 42-43 for further discussion of dendrimer synthesis andnomenclature.

Hyperbranched polymers which are dendrimers with structural defects ofthis type can be used in this invention.

Hyperbranched polymers which are not dendrimers may, for example, beformed by polymerization of a reactive monomer or more than one reactivemonomer. For example hyperbranched polymers which are polyamines may beprepared by polymerization of aziridine for example by treatment withbase.

Exemplarly core moieties include amines such as the moiety of ammonia(Mw 14 Da), diamines (e.g. ethylene diamine (Mw 56 Da), propylenediamine (Mw 70 Da) or 1,4-diaminobutane (Mw 84 Da)), and triamines (e.g.diethylenetriamine (NCH₂CH₂NHCH₂CH₂N) (Mw 99 Da) or1,2,3-triaminopropane (89Da)). Other cores may be oxygen containingincluding C(Me)(CH₂O)₃ (Mw 117 Da) or sulfur containing including(NCH₂CH₂S—SCH₂CH₂N) (Mw 148 Da).

Cationic hyperbranched polymers will have a predominantly positivecharge at about pH 7 that is to say that they either contain onlyuncharged groups and charged groups having positive charge at pH 7 orelse (less preferred) have groups that are negatively charged at pH 7that are outnumbered by groups that are positively charged. Cationichyperbranched polymers of this invention typically will have primaryamines as functional end groups.

Hyperbranched polymers of use according to the invention may contain anumber of functionalities for example they may be polyamines (entirelyor substantially containing secondary and tertiary amine groups and withprimary amines as functional end groups), polyamidoamines (amide groupsand secondary and tertiary amine groups and with primary amines asfunctional end groups) or polyethers with amine functionality (e.g.polyethers such as PEGs in which end groups have been transformed intoprimary amine groups).

An exemplary family of hyperbranched polymers are the polyamidoamines(PAMAMs) in which a moeity of ammonia or a di- or tri-amine (e.g.ethylenediamine) may be used as the core moiety and the addition ofgenerations of the branched molecule may be built up by reacting theammonia or the free amine groups with e.g. methyl acrylate followed byethylene diamine leading to a structure having a number of free aminegroups on the outer surface. Subsequent generations can be built up byfurther reaction with methyl acrylate and ethylene diamine. A structurein which all primary amine groups of the inner layers have been reactedwith methyl acrylate and ethylenediamine will be a dendrimer. PAMAMdendrimers are available under the trade mark Starburst®, manufacturedby Dendritech Inc. Starburst dendrimers are sold by Dendritech Inc.,Sigma Aldrich and Dendritic Nanotechnologies (DNT).

Other exemplary hyperbranched polymers may include polyamines such aspolypropyleneimine (PPI) and polyethyleneimine (PEI) polymers formed bypolymerization of the respective building blocks. Hyperbranched polymersbased on PPI may also be synthesized from a core such as diaminobutaneand built up by reaction of the primary amine groups with acrylonitrilefollowed by hydrogenation. PPI dendrimers are available under the trademark Astramol™ and provided by DSM and Sigma Aldrich. Polyethyleneimine(PEI) polymers are available from e.g. BASF, Nippon Shokubai and WuhanBright Chemical.

Thus, the hyperbranched polymer may be selected from polyamidoamine,polypropyleneimine, polyethyleneimine and other polyamine polymers andcopolymers comprising one or more of polyamidoamine, polypropyleneimine,polyethyleneimine and polyamine hyperbranched polymers.

In general, cationic hyperbranched polymers having primary amine groupsas functional end groups, for example PAMAMs or polyethylenimines orpolypropyleneimines, are particularly suitable for use in the presentinvention.

Hyperbranched aminated polymers comprising esters, carbonates,anhydrides and polyurethanes are less suitable as they tend to degrade.However, the biostability can depend on the number and proportion ofbiodegradable groups and some may therefore be suitable within thisinvention.

The properties of certain hyperbranched polymers are described in Table1 below:

TABLE 1 Examples of hyperbranched polymers with appropriate ratio oftotal molecular weight to core moiety molecular weight Molecular TypeSupplier Brand name Core weight [Da] Ratio PEI BASF Lupasol ®Ethane-1,2- 25,000  ~450:1 WF diamine (Mw 56 Da) PAMAM DendritechStarburst ® Ethane-1,2- 7,000-935,000 ~125:1-16,700:1 DNT G3-G10 diamine(e.g. 7,000-900,000) Sigma Aldrich (Mw 56 Da) PPI DSM Astramol ™Butane-1,4- 7,000  ~85:1 Sigma Aldrich Am-64 diamine (Mw 84 Da) PEINippon Epomin-P- Ethane-1,2- 70,000 ~1250:1 Shokubai 1050 diamine (Mw 56Da) PEI Wuhan Bright G-35 Ethane-1,2- 70,000 ~1250:1 Chemical diamine(Mw 56 Da) Examples of polymers with other types of structure (notsuitable hyperbranched polymers within the terms of the presentinvention) Molecular Type Manufacturer Brand name Core weight [Da] RatioPEI BASF Lupasol ® Undefined, 1,000,000 N/A SN polymeric PEI BASFLupasol ® Undefined, 2,000,000 N/A SK polymeric PEI Wuhan Bright G-35Ethane-1,2- 1,500  ~25:1 Chemical diamine (Mw 56 Da) PAMAM DendritechStarburst ® Ethane-1,2- 1,430  ~26:1 DNT G1 diamine Sigma Aldrich (Mw 56Da) PPI DSM Astramol ™ Butane-1,4- 316   ~4:1 Am-8 diamine (Mw 84 Da)

The PAMAM illustrated in FIG. 2 is based on ethylenediamine as coremoiety. The properties according to the number of generations built upare described in Table 2 below:

TABLE 2 Measured diameter/ Number of Ratio of Total Generation Mw (Da)Angstrom surface groups Mw to core Mw Core/G0 56/517* 15 4    ~9:1 11,430 22 8   ~26:1 2 3,256 29 16   ~58:1 3 6,909 36 32   ~125:1 4 14,21545 64   ~250:1 5 28,826 54 128   ~515:1 6 58,048 67 256 ~1,040:1 7116,493 81 512 ~2,080:1 8 233,383 97 1,024 ~4,170:1 9 467,162 114 2,048~8,340:1 10  934,720 135 4,096 ~16,700:1  See Aldrichimica Acta (2004)37(2) 1-52 “Dendrimers: building blocks for nanoscale synthesis”*Structure, see Scheme 1

Synthesis of an exemplary PEI hyperbranched polymer based onethylenediamine core by polymerization of aziridine is shown in Scheme2.

Synthesis of an exemplary PPI dendrimer based on butane,1,4-diamine coreby polymerization of acrylonitrile is shown in Scheme 3.

The hyperbranched polymer molecules useful in the present inventiontypically have a molecular weight of about 1,500 to 1,000,000 Da, moretypically about 10,000 to 300,000 Da e.g. about 25,000 to 200,000 Da.The hyperbranched polymer molecules useful in the present inventionsuitably are substantially spherical in shape. Typically they have adiameter of about 2 to 100 nm, e.g. 2 to 30 nm, especially about 5 to 30nm as determined by laser light scattering.

When the hyperbranched polymer is a PAMAM dendrimer, it typically has amolecular weight of about 5,000 to 1,000,000 Da, more typically about12,000 to 125,000 Da and a diameter of about 1 to 20 nm, e.g. 2 to 10nm,especially about 4 to 9 nm.

In hyperbranched polymers of use according to the invention the ratio oftotal molecular weight to core moiety molecular weight is at least 80:1,for example at least 100:1, for example at least 200:1 e.g. at least500:1 e.g. at least 1000:1. The ratio is typically less than 20,000:1e.g. less than 10,000:1 e.g. less than 5,000:1. For example the ratio isbetween 80:1 and 20,000:1 e.g. 200:1 and 5,000:1 e.g. between 200:1 and1600: 1 e.g. between 400:1 and 1600:1.

For the avoidance of doubt, the total molecular weight of thehyperbranched polymer referred to herein excludes the weight of anycovalently attached anti-coagulant entity or any beneficial agent.

The ratio is dictated by the molecular weight of the core and the totalmolecular weight of the hyperbranched polymer. The calculated ratio willvary as the core varies (in terms of chemical composition and molecularweight) and as the molecular weight of the generations varies (in termsof molecular weight of monomers and number of monomers attached in eachgeneration).

For PAMAM dendrimers a core derived from ethane-1,2-diamine is preferredand the number of generations is preferably between 3 and 10, morepreferably between 4 and 7 i.e. 4, 5, 6 or 7.

For PAMAM hyperbranched polymers, a core derived from ethylenediamine ispreferred and the number of incorporated reactive monomers(methylacrylate, Mw=56 Da and ethylenediamine, Mw=57 Da) in thehyperbranched polymer is exemplarily between 50 and 9,000 e.g between100 and 5,000 e.g. between 100 and 2,000 of each monomer.

For PEI hyperbranched polymers, a core derived from ethylenediamine ispreferred and the number of incorporated aziridine monomers (Mw=42 Da)in the hyperbranched polymer is exemplarily between 110 and 20,000 e.gbetween 110 and 10,000 e.g. between 110 and 3,000 monomers.

For PPI hyperbranched polymers, a core derived from butane-1,4-diamineis preferred and the number of incorporated acrylonitrile monomers(Mw=56 Da) in the hyperbranched polymer is exemplarily between 120 and17,000 e.g between 120 and 4,000 e.g. between 120 and 1,000 monomers.

In the device of the present invention, the plurality of cationichyperbranched polymer molecules may optionally be cross-linked to oneanother on the surface of the device. Cross-linking may take placeeither before or after the hyperbranched polymer molecules are appliedto the surface of the device and either before or after theanti-coagulant entities are attached thereto (see FIGS. 5, 6).

In the case where the hyperbranched polymer molecules are cross-linked,the number of molecules that may be cross-linked to form an aggregatehyperbranched polymer is two or more and, for example, from 2-500 e.g.from 2-10 such as from 2-5; and each molecule may be attached to anothermolecule in the aggregate by one or more cross-linkages e.g. up to 10cross linkages.

Aggregates of 2 or more hyperbranched polymer molecules useful in thepresent invention typically have a molecular weight of about 3,000 to2,000,000 Da, more typically about 50.000 to 500,000 Da. Thehyperbranched polymer aggregates useful in the present inventiontypically have a diameter of about 5 to 100 nm, especially about 20 to100 nm.

Derivatisation of Hyperbranched Polymer Molecules With Anti-coagulantEntities

Hyperbranched polymer molecules have a large number of functional endgroups which can be reacted with anti-coagulant entities such as heparin(see FIG. 4). The functional end groups can be of the same type or ofseveral different types, as appropriate. Therefore, one of theadvantages of the present invention is that it is possible to design themolecule such that it has a required number of functional end groups ofa specific functionality. This makes it possible to selectivelyimmobilize the desired amount of anti-coagulant entities on the surfaceof a device without interfering with the build up of the underlyinglayers.

The branching structure of the hyperbranched molecules makes it possibleto obtain a higher surface density of anti-coagulant entities than waspossible using essentially linear polymer structures, while stillachieving sufficient spacing of those anti-coagulant entities to ensurethat the bioavailability of each entity is not reduced in comparisonwith that achieved using previously known coatings and may actually beincreased.

Another useful feature of hyperbranched polymers is that the majority ofthe reactive functional end groups are on the surface of thehyperbranched molecule and therefore substantially all of theanti-coagulant entity is available on the surface of the hyperbranchedpolymer. The effect is particularly marked in the case of dendrimers,where all of the available functional groups are on the surface. Thisfeature gives a particular advantage over conventional coating polymersin which many of the reactive functional end groups may be hidden in theinterior of the structure rather than on the surface. This means thatanti-coagulant entity which reacts with functional groups in suchconventional coating polymers may be immobilized in the interstices ofthe polymer surface and will not be bioavailable.

The derivatised hyperbranched polymer architecture will allow a morehomogenous distribution of the anti-coagulant entity throughout thelayers in which it is incorporated, such as the outer coating layer,which should, in principle, result in increased ageing stability.Further, the possibility of selecting and adjusting the anti-coagulantdensity on the hyperbranched polymer will allow for a more robust andpredictable anti-coagulant distribution on the device. Thepre-fabrication of the hyperbranched polymer-anti-coagulant entityconjugate also allows a lower batch to batch variability, since it iseasier to adjust the degree of substitution of the hyperbranched polymerby the anti-coagulant entity (e.g. heparin) in solution rather than on asurface.

In a further aspect of the invention there is provided a cationichyperbranched polymer molecule characterized by having (i) a core moietyof molecular weight 14-1,000 Da (ii) a total molecular weight of 1,500to 1,000,000 Da (iii) a ratio of total molecular weight to core moietymolecular weight of at least 80:1 (e.g. at least 100:1) and (iv)functional end groups, whereby one or more of said functional end groupshave an anti-coagulant entity covalently attached thereto.

Depending on the number of anti-coagulant entities attached tofunctional end groups, and their charge (e.g. negatively charged in thecase of heparin as anti-coagulant entity), the cationic hyperbranchedpolymer may have a net positive or a net negative charge.

Suitably the anti-coagulant entity has a covalent connection only to asingle functional end group on one hyperbranched polymer molecule andnot to any other molecule. The coupling of the anti-coagulant entity isnever to the core of the hyperbranched polymer, only to a functional endgroup of the hyperbranched polymer.

The number of functional end groups which have an anti-coagulant entitycovalently attached thereto is one or more, for example 2 or more, forexample 2 to 200 e.g. 10 to 100 however there is no specific upperlimit. The number that may be attached will depend on the number of endgroups that are available, which is a function of the size of thecationic hyperbranched polymer molecule. The number of functional endgroups which have an anti-coagulant entity covalently attached theretomay for example be 1 to 95% e.g. 5 to 95% e.g. 10 to 80% e.g. 10 to 50%of available functional end groups. The number of functional end groupswhich have an anti-coagulant entity covalently attached thereto may forexample be 5 to 50% e.g. 5 to 40% e.g. 5 to 30% e.g. around 25% ofavailable functional end groups. When the anti-coagulant entities areanionic (for example, in the case of heparin moieties), the number thatmay be attached will also depend on whether it is desired for theresultant derivatised hyperbranched polymer to have a net positivecharge (in which case there should not be too many anionicanti-coagulant entities covalently attached) or a net negative charge.

Coupling of the Anti-coagulant Entity to the Cationic HyperbranchedPolymer

Typically, each anti-coagulant entity is covalently connected to acationic hyperbranched polymer via a linker and optionally one or morespacers. The linker is formed by the reaction of a functional end groupon the hyperbranched polymer with a functional group on theanti-coagulant entity. Table 3 and Scheme 4 show examples of some typesof linkers suitable for attaching the anti-coagulant entity to thehyperbranched polymer along with the functional groups from which thecovalent linker is formed and the type of reaction used. See e.g.reference (ISBN: 978-0-12-370501-3, Bioconjugate techniques, 2^(nd) ed.2008). However, radical coupling reactions may also be contemplated.

For each linker, one of the functional end groups is on thehyperbranched polymer and the other is on the anti-coagulant entity. Inprinciple, either way round is possible i.e. by reference to Table 3,functional groups 1 and 2 may respectively be on the hyperbranchedpolymer and on the anti-coagulant entity or may respectively be on theanti-coagulant entity and on the hyperbranched polymer.

In some cases, the anti-coagulant entity and the hyperbranched polymermay be joined by a linker which comprises more than one functionalgroup. For example, in the case where the linker is a thioether, abifunctional molecule (having, for example an SH group at each end) canbe connected at each end, respectively, to an alkyne/alkenefunctionalized anti-coagulant entity and an alkyne/alkene functionalizedhyperbranched polymer molecule resulting in the linker containing twothioethers. Alternatively, a bis-alkyne/alkene molecule can be connectedat each end, respectively, to a thiol functionalized anti-coagulantentity and a thiol functionalized hyperbranched polymer also resultingin the linker containing two thioethers. Similar possibilities exist forother linker types, as is clear from Table 3. The hyperbranched polymermay also carry two or more different functional groups, for exampleamine and alkyne functionality, so that anti-coagulant entities may beattached to the functional end groups of the hyperbranched polymer viamore than one type of linker, however, we prefer attachinganti-coagulant entity using one type of linker.

The linker moiety will typically have a molecular weight of around 14 to200 e.g. 14 to 100 Da.

TABLE 3 Type of Func. Func. reaction group 1 group 2 Linker Reductiveamination

Amidation

Michael addition

Michael addition

Thiol-Ene Click

Thio- Bromo

Thiol-Yne Click

CuAAC Click

Amidation (NHS-activated)

Amidation/ Disulfide (SPDP)

Illustrative chemistries shown in Table 3 and Scheme 4 are discussedbelow:

—C—NH—C— Linkage

Reductive amination: A reductive amination, also known as reductivealkylation, is a form of amination that involves the conversion of acarbonyl group to an amine linker via an intermediate imine (Schiff'sbase). The carbonyl group is most commonly a ketone or an aldehyde.

—C—NH—CHR—CHR—C(═O)— Linkage

Michael addition: The Michael reaction or Michael addition is thenucleophilic addition of a carbanion or another nucleophile (e.g.primary amine or thiol) to an alpha, beta unsaturated carbonyl compound.It belongs to the larger class of conjugate additions. This is one ofthe most useful methods for the mild formation of C—C bonds.

—C—S—C— Linkage

Thio-bromo: Thioether linkages are typically prepared by the alkylationof thiols. Thiols may react with bromide compounds to generate thioetherlinkages. Such reactions are usually conducted in the presence of base,which converts the thiol into the more nucleophilic thiolate.

Thiol-Ene and Thiol-Yne: Alternatively, thioether linkages may beprepared by reaction of a first compound containing a thiol group with asecond compound containing an alkene or an alkyne group. The first andsecond compounds can each be the hyperbranched polymer molecule and theanti-coagulant entity as appropriate.

Suitably the reaction takes place in the presence of a reducing agentsuch as tris(2-carboxyethyl)phosphine hydrochloride, or alternativelydithiothreitol or sodium borohydride, to avoid or reverse the effectiveof undesirable coupling of two thiol groups through oxidation.

In one embodiment the reaction is initiated with a radical initiator. Anexample of a radical initiator is 4,4′-azobis(4-cyanovaleric acid).Further examples are potassium persulfate,2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride,azobisisobutyronitrile (AIBN),1,2-bis(2-(4,5-dihydro-1H-imidazol-2-yl)propan-2-yl)diazenedihydrochloride,2,2′-(diazene-1,2-diyl)bis(2-methyl-1-(pyrrolidin-1-yl)propan-1-imine)dihydrochloride,3,3′-((diazene-1,2-diylbis(1-imino-2-methylpropane-2,1-diyl))bis(azanediyl))dipropanoicacid tetrahydrate, benzophenone and derivatives of benzophenone such as4-(trimethyl ammoniummethyl) benzophenone chloride. A further example isammonium persulfate.

In another embodiment, the reaction is not initiated with a radicalinitiator. Instead, conditions of higher pH (e.g. pH 8-11) are used.This type of reaction is more suitable when an activated alkene oralkyne is used for reaction with the thiol.

The reaction between a first compound containing a thiol group and asecond compound containing an alkyne group may be represented asfollows:

-   where one of R^(a) and R^(b) is the hyperbranched polyamine and the    other of R^(a) and R^(b) is the anti-coagulant entity.

When an alkene containing linker is formed, this compound may undergo afurther chemical transformation with e.g. a thiol (as shown in Table 3)or an amine.

Where the second compound is derivatised with an alkene, in oneembodiment an activated alkene is used. An example of a suitableactivated alkene is a maleimide derivative.

The reaction between a first compound containing a thiol group and asecond compound containing a maleimide group may be represented asfollows:

-   where one of R^(a) and R^(b) is the polymer and the other of R^(a)    and R^(b) is the anti-coagulant entity. The reaction is generally    carried out in the presence of tris(2-carboxyethyl)phosphine    hydrochloride as reducing agent, and 4,4′-azobis(4-cyanovaleric    acid) as radical initiator, and under acidic conditions.    Triazole Linkage (CuAAC Coupling)

Azide-Alkyne: 1,2,3-triazole linkages may be prepared by reaction of analkyne and an azido compound. The reaction to form the linker may bebetween an alkyne group on one of the anti-coagulant entity and thehyperbranched polymer molecule and an azido group on the other of theanti-coagulant entity and the hyperbranched polymer molecule. Methodsfor carrying out this reaction are similar to the methods described inWO 2010/029189.

The reaction between the azide and the alkyne groups may be carried outat elevated temperatures (T>60° C.) or in the presence of a metalcatalyst, for example a copper, e.g. a Cu(I) catalyst using reactionconditions conventionally used in the Huisgen cycloaddition (the1,3-dipolar cycloaddition of an azide and a terminal alkyne to form a1,2,3-triazole). The Cu(I) catalyst may, if desired, be produced insitu, e.g. by reduction of a corresponding Cu(II) compound for exampleusing sodium ascorbate. The reaction may also, if desired, be carriedout under flow conditions.

The CuAAC reaction may, for example be carried out at a temperature offrom about 5 to 80° C., preferably at about room temperature. The pHused in the reaction may be from about 2-12, preferably about 4-9 andmost preferably at about 7. Suitable solvents include those in which theentity attached to the azide or alkyne is soluble, e.gdimethylsulfoxide, dimethylformamide, tetrahydrofuran and preferablywater or mixtures of water with one of the above. The proportion of theentity to the surface may be adjusted to provide the desired density ofthe entity on the surface.

—C(═O)—N— Linkage

Amidation: Amides are commonly formed via reactions of a carboxylic acidwith an amine. Carboxylic acids and carboxylic acid derivatives mayundergo many chemical transformations, usually through an attack on thecarbonyl breaking the carbonyl double bond and forming a tetrahedralintermediate. Thiols, alcohols and amines are all known to serve asnucleophiles. Amides are less reactive under physiological conditionsthan esters.

Amidation using activated acid: Activated acids (basically esters with agood leaving group e.g. NHS-activated acids) can react with amines toform amide linkers, under conditions where a normal carboxylic acidwould just form a salt.

—C—S—S—CH₂—CH₂—C(═O)—N— Linkage

Coupling using SPDP reagents: The N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and its analogues belong to a unique group of amine-and thiol-reactive heterobifunctional link forming reagents that producedisulfide-containing linkages.

Reductive amination, Michael addition, thio-bromo reactions, amidationusing NHS-activated acid, coupling using SPDP reagent, CuAAC andthiol-ene couplings are all suitable to provide benign couplingconditions and high yield of linker formation.

The groupings shown in Table 3 are for illustrative purposes only andalternative or variant functionalities may of course be employed. Forexample, the amine groups may be positioned on a secondary carbon or thealiphatic chains illustrated may be replaced by aromatic groups.

Free Radical Initiated Reactions

As mentioned briefly above, the functional end groups of thehyperbranched polymer molecule may be coupled to an anti-coagulantentity by a linker formed through a free radical initiated reaction.Radicals may be created for example via heat, photolysis (e.g. Norrishtype I and/or Norrish type II reactions), ionization, oxidation, plasmaor electrochemical reactions For example when a hyperbranched polymermolecule that has free primary amine groups is treated withbenzophenone, radicals such a e.g. carbon or oxygen radicals are createdwhich may participate in free radical initiated reactions (such asreaction with alkenes).

In one embodiment the covalent linker comprises a secondary aminelinkage. In particular, the linker may comprise a —NH— group;

In another embodiment, the covalent linker comprises an amide linkage.In particular, the linker may comprise a —NH—C(O)— group;

In another embodiment the covalent linker comprises a thioether linkage.

In another embodiment, the covalent linker comprises a 1,2,3-triazolelinkage.

The term “thioether linkage” refers to a connection between a sulfur andtwo carbon atoms. This connection is sometimes referred to as “sulfide”.The sulfur may be attached to two saturated carbon atoms (i.e. —C—S—C—)or it may be attached to a saturated and an unsaturated carbon atom(i.e. —C—S—C═).

The term “thiol” refers to an —S—H moiety.

The term “secondary amine linkage” refers to a connection between an NHgroup and two carbon atoms, i.e. —C—NH—C—.

The term “amide linkage” refers to a connection between two carbon atomsof the type —C—C(O)NH—C—.

In one embodiment, the linker between the anti-coagulant entity such asa heparin moiety and a functional end group of the hyperbranched polymermolecule is an unbranched linker.

The linker can be biodegradable or non-biodegradable but is moresuitably non-biodegradable in order that a coated device isnon-thrombogenic for a long period of time.

Where there is a multiplicity of linkers it is possible for some or allof them to be of a different type.

In one embodiment, all of the linkers are of the same type.

Spacers

At its simplest the covalent connection between the functional end groupof the hyperbranched polymer molecule and the anti-coagulant entity isvia a linker e.g. as shown in Table 3. However optionally the linker maybe separated by a spacer from either the surface or the anti-coagulantmoiety or both.

The purpose of the spacer, if employed, is usually to significantlyincrease the separation between the hyperbranched polymer molecule andthe anti-coagulant entity i.e. in effect to significantly increase theseparation between the surface of the device and the anti-coagulantentity. For example, the molecular weight of the spacer may be from 50to 10⁶ Da, typically 100 to 10⁶ Da e.g. 100 to 10⁴ Da. The length of thespacer may for example be from 10 to 10³ Å. We prefer the spacer to bestraight chain. In some embodiments the spacer is hydrophilic, forexample, it may comprise a PEG chain. In one aspect, the covalentconnection between the functional end group of the hyperbranched polymermolecule and the anti-coagulant entity may be viewed as having threeportions—“spacer A” between the functional end group of thehyperbranched polymer molecule and the linker moiety, the linker moiety,and “spacer B” between the linker moiety and the entity. In oneembodiment the molecular weight of spacer A is between 50 and 10³ Da. Inanother embodiment the molecular weight of spacer B is between 50 and10³ Da. In one embodiment spacer A comprises one or more aromatic rings.In another embodiment spacer A does not comprise any aromatic rings. Inone embodiment spacer B comprises one or more aromatic rings. In anotherembodiment spacer B does not comprise any aromatic rings. In oneembodiment spacer A is hydrophilic. In another embodiment spacer B ishydrophilic. In one embodiment spacer A comprises a PEG chain. Inanother embodiment spacer B comprises a PEG chain. In one embodimentspacers A and B are both hydrophilic, for example they each comprise aPEG chain. As used herein, a PEG chain refers to a polymeric chainobtainable by polymerisation of ethylene oxide, typically of weightbetween 100 and 10⁶ Da. In another aspect, the covalent connection maycomprise two or more triazole rings. In another embodiment, the covalentconnection may be viewed as having five portions—“spacer A” between thesurface and a first linker moiety, the first linker moiety, “spacer B”between the first linker moiety and a second linker moiety, the secondlinker moiety, and “spacer C” between the second linker moiety and theentity. In one embodiment the molecular weight of spacer A is between 50and 10³ Da. In one embodiment the molecular weight of spacer B isbetween 100 and 10⁶ Da. In one embodiment the molecular weight of spacerC is between 50 and 10³ Da. In one embodiment spacer A and/or spacer Band/or spacer C is hydrophilic for example comprising a PEG chain. Forexample spacer B (at least) may comprise a PEG chain.

Although spacers may be present they are typically not necessary sinceit should be noted that the structure of the hyperbranched polymers, byvirtue of their size and shape, provides for some separation of theanti-coagulant entity from the surface of the device.

In cases where spacers are present, they are for example straight chainspacers of about 10 to 10³ Å.

A specific merit of having a spacer that comprises a PEG chain (or otherhydrophilic polymer) is to provide the device with lubriciousproperties.

The spacer can be biodegradable or non-biodegradable but is moresuitably non-biodegradable in order that a coated device isnon-thrombogenic for a long period of time (i.e. the coated device haspreserved non-thromogenic properties).

Functionalization of Coating Building Blocks

-   i. Linker formation where no prior modification of hyperbranched    polymer molecule or anti-coagulant entity is required

Several of the linkers shown above in Table 3 can be formed directly bythe reaction of a functional end group of a hyperbranched polymer, forexample a hyperbranched polyamine with an anti-coagulant entitycontaining an aldehyde.

Thus, the reductive amination, the Michael addition, the SPDP reactionand the amidation reactions shown in Table 3 require the presence of aprimary amine functional end group. Hyperbranched molecules such ashyperbranched polyamines e.g.PAMAM dendrimers possess suitable freeprimary amine groups for use in these linkage forming reactions andtherefore do not require further modification.

Therefore, in one embodiment, the hyperbranched polymer molecule carriesmultiple free primary amine groups as functional end groups and is, forexample, a PAMAM, PPI or PEI hyperbranched polymer molecule.

Nitrous acid degraded heparin and native heparin bear reactive groups,an aldehyde group and a hemi-acetal function respectively, at theirreducing end and thus nitrous acid degraded heparin or native heparincan be reacted with a hyperbranched polymer having free primary aminegroups in a reductive amination reaction to form a linker containing asecondary amine group as shown in Table 3 and Scheme 4 above.

Methods of forming a secondary amine linkage between an aminefunctionalized surface and a heparin derivative are described, forexample in EP-B-0086186, EP-B-0086187, EP-B-0495820 and U.S. Pat. No.6,461,665.

-   ii. Linkage formation where modification of hyperbranched polymer    and/or anti-coagulant entity is required

Alternatively, either or both of the anti-coagulant entity and thehyperbranched polymer may be modified to carry a suitable functionalgroup as will be discussed in greater detail below.

Methods of derivatising heparin and other anti-coagulant entities e.g.to incorporate alkyne and azide groups are disclosed in WO2010/029189the contents of which are herein incorporated by reference in theirentirety.

Therefore, for some of the linkers described above in Table 3, it isnecessary to pre-prepare functionalized derivatives of either or both ofthe hyperbranched polymer molecule and the anti-coagulant entity.

The hyperbranched polymer molecule may be functionalized usingtechniques known in the art. Primary amino groups on a PAMAM dendrimeror similar hyperbranched polymer may be used as points of attachment fora suitable functional group for forming the chosen covalent linkage, forexample an alkene, alkyne, thiol, halo or azido group. Hencehyperbranched polyamines may be functionalized to bear alkene, alkyne,thiol, halo or azido groups by conventional means e.g. by reactingpendant primary amino groups on the polyamine with an activatedcarboxylic acid (e.g. an N-hydroxy succinimide derivative of acarboxylic acid) containing an alkene, alkyne, thiol, halo or azidogroup.

Thus, in order to introduce suitable alkene or alkyne groups, ahyperbranched polyamine molecule bearing a number of primary aminegroups represented as follows:R″—NH₂

-   where R″ is the hyperbranched polyamine residue;    may be reacted with a compound of the formula:

-   where n is an integer from 1 to 8 e.g. 1 to 4;    to give a maleimide functionalized polyamine of the formula:

-   where R″ and n are as defined above.

Alternatively, the hyperbranched polyamine may be reacted with anactivated alkyne-containing group of the formula:

-   where n is an integer from 1 to 8, e.g. 1 to 4;    to give an alkyne functionalized hyperbranched polyamine of the    formula:

-   where R″ and n are as defined above.

Similarly, a hyperbranched polymer having free primary amines asfunctional end groups may be derivatised with a thiol group. In thiscase, a hyperbranched polyamine such as a PAMAM dendrimer bearing anumber of primary amine groups represented as follows:R″—NH₂

-   where R″ is as defined above;    may be reacted with an thiol-containing activated carboxylic acid,    for example a compound of the formula:

-   where n is an integer from 1 to 8, e.g. 1 to 4;    to give a derivatised polymer of the formula:

-   where R″ and n are as defined above.

Halo groups may be introduced into the hyperbranched polymer molecule ina similar manner.

One may also consider using other amidation reactions involving SPDP or1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to obtain the samefunctionalization.

An anti-coagulant entity, e.g. heparin, carrying an alkene, aldehyde,alkyne, thiol, azo, amine, halide, activated carboxylic acid, maleimideester or an α,β-unsaturated carbonyl group may be made by conventionalmethods known per se. For example an anti-coagulant entity, e.g.heparin, carrying an alkyne/alkene group may be made by the reaction ofan alkoxyamine of the formula:R¹—O—NH₂wherein R¹ is an alkyne/alkene-containing group; with an aldehyde orhemi-acetal group on the anti-coagulant entity using conventionaltechniques known per se, see e.g. Example 3a, 3b and 3c ofWO2010/029189. This type of reaction proceeds via formation of anoxy-imine function to give a compound of the formula:R¹—O—N=R′in which R¹ is as defined above and R′ is the residue of theanti-coagulant entity.

Nitrous acid degraded heparin and native heparin bear reactive groups,an aldehyde group and a hemi-acetal function respectively, at theirreducing end which may be linked in this way.

Similarly, an anti-coagulant entity derivatised with a thiol group maybe formed by the reaction of an aldehyde or hemi-acetal group on theanti-coagulant entity with a compound of the formula:HS—X—NH₂where X is a hydrocarbon spacer, for example (CH₂)_(n) where n is 1 to 8e.g. 1 to 4, in which one or more (e.g. 1 or 2) methylene groups areoptionally replaced by O; or X comprises a PEG chain containing 1 to 100(e.g.1 to 50 such as 1 to 10) ethylene glycol units;to give a product of the formulaR′—CH₂—NH—X—SHwhere X is as defined above and R′—CH₂— is the residue of theanti-coagulant entity.

An example of such a procedure is given in Example 4.2 below.

A similar method can be employed for the introduction of an azido groupor a halo group such as fluoro, chloro or bromo.

As discussed above, one reason to modify the hyperbranched polymer is tointroduce certain functional groups to permit coupling to theanti-coagulant entity. When the hyperbranched polymer has certainexisting functional end groups e.g. primary amine groups, these may beconverted to other functional groups, for example, azide or alkynegroups. All or (more usually) some (e.g. 0.5 to 25%) of the functionalgroups may be converted for this purpose.

It may also be desired to introduce new functional groups for otherpurposes. For example some (e.g. 0.5 to 25%) existing functional endgroups (e.g. primary amine groups) may be converted to other functionalgroups, for example, azide or alkyne groups to permit attachment ofbeneficial agents e.g. lubricious agents mentioned below.

Surface Coating

The device has a surface comprising a layered coating formed of one ormore layers. The device, especially when it is a medical device, mayhave one or more portions containing void spaces, or pores. The poresmay be within the device and/or be part of at least one surface of thedevice. An example of a porous medical device is expandedpolytetrafluoroethylene (ePTFE). The pores may have a coating layer ornot.

Desirably a portion of the surface (desired to be non-thrombogenic) orthe whole of the surface of the device is covered with a coating.

The surface of a device may have one or many coating layers (e.g. 2 ormore, or 3 or 4 or 5 e.g. up to 20 coating layers) and the term “outercoating layer” refers to a coating layer which, in a medical device, isin contact with the tissues of the patient or is in contact with bodyfluids, or in an analytical or separation device, comes into contactwith a substance to be analysed, separated or handled. Thus, the outercoating layer may be the coating layer on the outer and/or the innersurface of a hollow device or a device of open structure such as astent. A layer which is not the outer coating layer is referred toherein as an “underlayer”.

According to the invention the outer coating layer comprises a pluralityof cationic hyperbranched polymer molecules to which are covalentlyattached via functional end groups one or more anti-coagulant entities.

In general, the majority, or even all, of the cationic hyperbranchedpolymer molecules of the outer coating layer will have a plurality ofanti-coagulant entities covalently attached thereto via their functionalend groups.

The optimum number of layers will depend on the type of material fromwhich the device is made, and the contemplated use of the surfacecoating. The number and nature of the layers needed to provide a fullcoverage of the device surface can be easily determined by those skilledin the art. The surface coating may, if desired, be made up layer bylayer.

For example, the coating layer(s) may be formed by adsorbing on thesurface of the device a cationic polymer, followed by the application ofa solution of an anionic polymer, e.g. an anionic polysaccharide, e.g.dextran sulfate or a functionalized cationic hyperbranched polymer witha net negative charge, to obtain at least one adsorbed layer of theanionic polymer. See Multilayer Thin Films ISBN: 978-3-527-30440-0.Hence the surface may comprise a layer of cationic polymer and a layerof anionic polymer e.g. a polysaccharide or a functionalized cationichyperbranched polymer with a net negative charge. More generally, thesurface coating may comprise one or more coating bilayers of cationicpolymer and anionic polymer. Typically the innermost layer (i.e. thelayer applied to the bare device surface, for example a metal, plasticor ceramic surface) is a layer of cationic polymer.

As discussed in more detail below, the outer coating layer comprising aplurality of cationic hyperbranched polymer molecules to which arecovalently attached one or more anti-coagulant entities via theirfunctional end groups may be applied in one of two ways. According tothe first way, cationic hyperbranched polymer molecules with a generalpositive charge may be applied to an anionic polymer on the surface ofthe device. The hyperbranched polymer molecules are then modified tolink them to the anti-coagulant entities. According to the second way,cationic hyperbranched polymer molecules to which are covalentlyattached one or more anti-coagulant entities via their functional endgroups may be applied to an anionic or cationic polymer on the surfaceof the device depending on whether the functionalised hyperbranchedpolymer molecules bear an overall positive or negative charge.

In some cases, the cationic hyperbranched polymer molecules may becross-linked to the polymer surface coating via reactive functionalgroups. If the cationic hyperbranched polymer is cross-linked to thedevice surface or to underlying coating layers before reaction with theanti-coagulant entity, it is necessary to ensure that a sufficientnumber of amino groups (or other reactive groups introduced) remainavailable to be able to link the desired amount of anti-coagulant entityto the outer coating layer. Alternatively, the cationic hyperbranchedpolymer molecules can be reacted with the anti-coagulant entities beforeapplication to the surface of the device or to a coating layer and thencross-linked. Typically there is no cross-linking directly between theanti-coagulant entity and the surface coating.

A range of cationic polymers may be used for the underlayers. Anexemplary cationic polymer is a polyamine (e.g. that described in EP0086187 Larsson and Gölander). Such polymers may be a straight chain butis more usually a branched chain polymer or alternatively ahyperbranched polymer, optionally cross-linked. Alternatively, one ormore (e.g. all of) the cationic polymer layers other than the outercoating layer may comprise (e.g. be formed of) cationic hyperbranchedpolymer molecules, which are the same as or similar to those used in theouter coating layer. Optionally these may also be cross-linked.

The coating procedure may be performed essentially as described inEP-B-0495820 and in this case it is only the outer coating layer whichcomprises the anti-coagulant entity.

The procedure of EP-B-0495820 may however be modified so that the outerlayer is the anionic polymer which is then coupled, as described below,with a cationic hyperbranched polymer to which is attached one or moreanti-coagulant entities (but that still retains a net positive charge)or is coupled with a cationic hyperbranched polymer with functional endgroup(s) capable of reacting with functional groups on an anti-coagulantentity to form a covalent linker moiety as described above.

According to one embodiment, there is provided a device wherein one ormore of the layers of the layered coating other than the outer coatinglayer comprises cationic hyperbranched polymer molecules characterizedby having (i) a core moiety of molecular weight 14-1,000 Da (ii) a totalmolecular weight of 1,500 to 1,000,000 Da (iii) a ratio of totalmolecular weight to core moiety molecular weight of at least 80:1 (e.g.at least 100:1) and (iv) functional end groups which are optionallyderivatised e.g. with one or more anti-coagulant entities.

According to one embodiment of the invention when underlayers comprisecationic polymers they may comprise cationic hyperbranched polymermolecules characterized by having (i) a core moiety of molecular weight14-1,000 Da (ii) a total molecular weight of 1,500 to 1,000,000 Da (iii)a ratio of total molecular weight to core moiety molecular weight of atleast 80:1 (e.g. at least 100:1) and (iv) functional end groups.According to this embodiment, these cationic hyperbranched polymermolecules may be the same as those used in the outer coating layer (butwithout the anti-coagulant entity attached) or they may be differenthyperbranched polymer molecules. In any event exemplary cationichyperbranched polymer molecules include those described elsewhere hereinin relation to those cationic hyperbranched polymer molecules that maybe used in preparation of the outer coating layer.

For example, all the underlayers which comprise cationic polymers maycomprise cationic hyperbranched polymer molecules characterized byhaving (i) a core moiety of molecular weight 14-1,000 Da (ii) a totalmolecular weight of 1,500 to 1,000,000 Da (iii) a ratio of totalmolecular weight to core moiety molecular weight of at least 80:1 (e.g.at least 100:1); and (iv) functional end groups.

The anionic polymer may also be a functionalized cationic hyperbranchedpolymer with a net negative charge.

According to one embodiment, when underlayers comprise anionic polymersthey may comprise cationic hyperbranched polymer molecules characterizedby having (i) a core moiety of molecular weight 14-1,000 Da (ii) a totalmolecular weight of 1,500 to 1,000,000 Da (iii) a ratio of totalmolecular weight to core moiety molecular weight of at least 80:1 (e.g.at least 100:1) and (iv) functional end groups, whereby one or more ofsaid functional end groups have an anionic anti-coagulant entitycovalently attached thereto thereby conferring on the molecules a netnegative charge.

For example, all the underlayers which comprise anionic polymers maycomprise cationic hyperbranched polymer molecules characterized byhaving (i) a core moiety of molecular weight 14-1,000 Da (ii) a totalmolecular weight of 1,500 to 1,000,000 Da (iii) a ratio of totalmolecular weight to core moiety molecular weight of at least 80:1 (e.g.at least 100:1) and (iv) functional end groups, whereby one or more ofsaid functional end groups have an anionic anti-coagulant entitycovalently attached thereto thereby conferring on the molecules a netnegative charge.

According to one embodiment, the layers of the coating on the surface ofthe device are all either (a) cationic hyperbranched polymer moleculescharacterized by having (i) a core moiety of molecular weight 14-1,000Da (ii) a total molecular weight of 1,500 to 1,000,000 Da (iii) a ratioof total molecular weight to core moiety molecular weight of at least80:1 (e.g. at least 100:1) and (iv) functional end groups or (b)cationic hyperbranched polymer molecules characterized by having (i) acore moiety of molecular weight 14-1,000 Da (ii) a total molecularweight of 1,500 to 1,000,000 Da and (iii) a ratio of total molecularweight to core moiety molecular weight of at least 80:1 (e.g. at least100:1) and (iv) functional end groups, whereby one or more of saidfunctional end groups have an anionic anti-coagulant entity covalentlyattached thereto thereby conferring on the molecules a net negativecharge.

One advantage of this is that the number of different components of thelayers of the coating is minimized.

Prior to applying the first coating layer (i.e. the innermost layer),the surface of the device may be cleaned to improve adhesion and surfacecoverage. Suitable cleaning agents include solvents as ethanol orisopropanol (IPA), solutions with high pH like solutions comprising amixture of an alcohol and an aqueous solution of a hydroxide compound(e.g. sodium hydroxide), sodium hydroxide solution as such, solutionscontaining tetramethyl ammonium hydroxide (TMAH), basic Piranha (ammoniaand hydrogen peroxide), acidic Piranha (a mixture of sulfuric acid andhydrogen peroxide), and other oxidizing agents including combinations ofsulfuric acid and potassium permanganate or different types ofperoxysulfuric acid or peroxydisulfuric acid solutions (also asammonium, sodium, and potassium salts).

Thus an aspect of the invention is a device having a surface coatingwherein the surface coating comprises one or more coating bilayers ofcationic polymer and anionic polymer, wherein the outer coating layer ofthe coating comprises a plurality of cationic hyperbranched polymermolecules characterized by having (i) a core moiety of molecular weight14-1,000 Da (ii) a total molecular weight of 1,500 to 1,000,000 Da (iii)a ratio of total molecular weight to core moiety molecular weight of atleast 80:1 (e.g. at least 100:1) and (iv) functional end groups, wherebyone or more of said functional end groups have an anti-coagulant entitycovalently attached thereto.

Formation of the Outer Coating Layer

As briefly described above, the heparin moiety or other anti-coagulantentity may be attached to the hyperbranched polymer molecules eitherbefore or after the hyperbranched polymer molecules are applied to thesurface of the device. The surface of the device to which the outercoating layer is applied may optionally contain one or more underlayers.See FIG. 7.

Therefore, in a further aspect of the invention there is provided aprocess for the manufacture of a device as described above, the processcomprising, in any order:

-   -   i. reacting a plurality of functional end groups of        hyperbranched polymer molecules with anti-coagulant entities        such that each hyperbranched polymer molecule is covalently        linked to a plurality of anti-coagulant entities; and    -   ii. attaching the hyperbranched polymer molecules to the surface        of a device.

As described above, the anti-coagulant entities are attached tohyperbranched polymer molecule via a covalent linkage and it may, insome cases, be necessary to carry out an additional step of modifyingthe hyperbranched polymer molecules and/or the anti-coagulant entitybefore step (i) in order to introduce suitable functional groups forforming a covalent linkage between the hyperbranched polymer moleculesand the anti-coagulant entity.

Suitable covalent linkages and methods for modifying the hyperbranchedpolymer and/or the anti-coagulant entity are discussed in detail above.As noted above, the linker may optionally be separated from the surfaceand/or the anti-coagulant moiety by a spacer. Thus the process mayoptionally involve the modification of the surface and/or theanti-coagulant moiety by provision of a spacer.

When the first step of the process above is step (i), the process ofattaching the anti-coagulant entities to the hyperbranched polymermolecules may be carried out in solution under appropriate reactionconditions with suitable solvents being, for example THF, DCM, DMF,DMSO, IPA, methanol, ethanol and water including mixtures thereof.

When the second step of the process is step (i) (i.e. the first step ofthe process is step (ii)), the outer coating layer of the device willusually be brought into contact with a solution of the anti-coagulantentity under the appropriate reaction conditions. Suitable solvents forthe anti-coagulant entity are, for example, IPA, ethanol, THF, DMF,DMSO, DCM and especially water including mixtures thereof.

In one embodiment, as already mentioned, two or more hyperbranchedpolymer molecules may be aggregated by cross-linking.

Therefore, the process above may further comprise the additional step ofcross-linking two or more hyperbranched polymer molecules to oneanother. The two or more hyperbranched polymer molecules may beaggregated by cross-linking before or after the hyperbranched polymermolecules are functionalized with the one or more anti-coagulantentities. The order in which cross-linking is performed may depend onthe device e.g. the geometry of the device. Preferably the cross-linkingis performed after the functionalisation. Furthermore, thiscross-linking step may take place either before or after the attachmentof the hyperbranched polymer molecules to the surface of the device.

The process may also include the step of cross-linking one or morehyperbranched polymer molecules to the surface of the device. Forexample hyperbranched polymer molecules to which are attached one ormore anti-coagulant entities on the outer coating layer may also becross linked to a cationic or anionic polymer of the layer underneaththe other coating layer.

This cross-linking step may be part of step (ii) above or, alternativelythe cross-linking step may be carried out after step (ii) in order tostrengthen the adhesion of the hyperbranched polymer molecules to thesurface of the device and enhance the stability of the coating.

If any required cross-linking, either between two or more hyperbranchedpolymer molecules or between hyperbranched polymer molecules and thesurface, is carried out before derivatisation, it is necessary to ensurethat sufficient free functional groups remain on the hyperbranchedmolecule to allow attachment of a suitable number of anti-coagulantentities. Alternatively, if derivatisation is carried out first, thenthe degree of derivatisation must be such that free functional groupsremain for any cross-linking that is required.

In general, it is preferred that step (i) is carried out before step(ii) since it is easier to control the amount of anti-coagulant entitywhich is attached to the hyperbranched polymer molecules and, inaddition, wastage of anti-coagulant entity is minimized, particularlywhen the reaction is carried out in solution as described above.

We provide as an aspect of the invention a device obtainable by theaforementioned processes.

Another aspect of the invention is a non-thrombogenic device which isobtainable by a process comprising:

-   -   (a) treating a device to present a surface coating comprising an        outer coating layer comprising cationic hyperbranched polymer        molecules characterized by having (i) a core moiety of molecular        weight 14-1,000 Da (ii) a total molecular weight of 1,500 to        1,000,000 Da and bearing functional end groups and (iii) a ratio        of total molecular weight to core moiety molecular weight of at        least 80:1 (e.g. at least 100:1);    -   (b) reacting one or more of said functional end groups with        molecules of an anti-coagulant entity which is functionalized to        bear groups which are capable of reacting with the reactive        functional groups on the hyperbranched cationic polymer;    -   thereby to attach the anti-coagulant entity to the hyperbranched        cationic polymer.

Another aspect of the invention is a non-thrombogenic device which isobtainable by a process comprising:

-   -   (a) treating a device to present a positively charged polymer        surface layer;    -   (b) associating with said polymer surface layer functionalized        cationic hyperbranched polymer molecules characterized by        having (i) a core moiety of molecular weight 14-1,000 Da (ii) a        total molecular weight of 1,500 to 1,000,000 Da and (iii) a        ratio of total molecular weight to core moiety molecular weight        of at least 80:1 (e.g. at least 100:1) and bearing a        multiplicity (e.g. 2 or 10 or 50 or 100 or 500 or more depending        on the number of available functional end groups) of negatively        charged anti-coagulant entities such as heparin moieties and        wherein said functionalized hyperbranched polymer has a net        negative charge.

Another aspect of the invention is a non-thrombogenic device which isobtainable by a process comprising:

-   -   (a) treating a device to present a negatively charged polymer        surface layer;    -   (b) associating with said polymer surface layer functionalized        cationic hyperbranched polymer molecules characterized by        having (i) a core moiety of molecular weight 14-1,000 Da (ii) a        total molecular weight of 1,500 to 1,000,000 Da and (iii) a        ratio of total molecular weight to core moiety molecular weight        of at least 80:1 (e.g. at least 100:1) and bearing one or more        negatively charged anti-coagulant entities such as heparin        moieties and wherein said functionalized hyperbranched polymer        has a net positive charge.

For example, the device is treated to present a surface comprising ananionic polymer for example a polysaccharide such as dextran sulfate,derivatives thereof or a functionalized cationic hyperbranched polymerwith a net negative charge.

Cross Linking

As described herein, hyperbranched polymer molecules of the outercoating layer may optionally be cross-linked to other hyperbranchedpolymer molecules of the outer coating layer or may be cross-linked tomolecules (e.g. hyperbranched polymer molecules) of an underlayer.Polymer molecules in underlayers may optionally be cross linked.

Suitably cross linking agents that may be used for these purposes willbe chosen according to the coupling chemistry required. Any di, tri, ormulti functional cross-linker may, in principle, be used such asfunctionalised PEGs and Jeffamines. For cross linking of amines it wouldbe suitable to use di-functional aldehydes such as crotonaldehyde orglutaraldehyde. In some cases epichlorohydrin may be useful.

Cross linking is capable of creating a covalent bond between afunctional end group of the hyperbranched polymer molecule of the outercoating layer and a functional end group of another hyperbranchedpolymer molecule of the outer coating layer or a molecule (e.g. ahyperbranched polymer molecule or a cationic or anionic polymermolecule) of an underlayer. Such cross-linking suitably does not involvethe anti-coagulant entity. Thus suitably the anti-coagulant entity has acovalent connection only to one hyperbranched polymer molecule and notto any other molecule. Suitably the cross linking of one hyperbranchedpolymer molecule to another hyperbranched polymer molecule involves useof functional end groups on the hyperbranched polymer molecule which arenot involved in linkage to the anti-coagulant entity. In one embodimentsaid functional groups used in cross-linking are formed byrefunctionalisation of the original functional end groups of thehyperbranched polymer molecule.

Devices

The device may be any device to which it is desirable to attachanti-coagulant entities, for example a medical device, an analyticaldevice or a separation device.

For the purposes of this patent application, the term “medical device”refers to implantable or non-implantable devices but more usually toimplantable medical devices. Examples of implantable medical deviceswhich may be permanent or temporary implantable medical devices includecatheters, stents including bifurcated stents, balloon expandablestents, self-expanding stents, stent-grafts including bifurcatedstent-grafts, grafts including vascular grafts, bifurcated grafts,artificial blood vessels, blood indwelling monitoring devices,artificial heart valves, pacemaker electrodes, guidewires, cardiacleads, cardiopulmonary bypass circuits, cannulae, plugs, drug deliverydevices, balloons, tissue patch devices and blood pumps.

Examples of non-implantable medical devices are extracorporeal devices,e.g. extracorporeal blood treatment devices, and transfusion devices.

Devices may have neurological, peripheral, cardiac, orthopedal, dermaland gynecological application, inter alia.

A medical device may have one or many coating layers and the term “outercoating layer” refers to a coating layer which, when the device isimplanted in a patient or is in use, is in contact with the tissues ofthe patient or is in contact with body fluids e.g blood. Thus, the outercoating layer may be the coating layer on the outer and/or the innersurface of a hollow device or a device of open structure such as astent.

An analytical device may be, for example, a solid support for carryingout an analytical process such as chromatography or an immunologicalassay, reactive chemistry or catalysis. Examples of such devices includeslides, beads, well plates, membranes etc. A separation device may be,for example, a solid support for carrying out a separation process suchas protein purification, affinity chromatography or ion exchange.Examples of such devices include filters and columns etc. Like a medicaldevice, an analytical or separation device may also have many coatinglayers and the term “outer coating layer” refers to a coating layerwhich comes into contact with a substance to be analysed, separated orhandled.

In some cases, it may be desirable to adjust the properties of thecoating and in this case one or more additional entities may be attachedto the hyperbranched polymer in addition to the anti-coagulant entity.For example, if it is desirable to increase the hydrophilicity of thehyperbranched polymer, the additional entities may comprise one or morePEG chains.

As used herein, the term “PEG chain” refers to a polymeric chainobtainable by polymerisation of ethylene oxide, typically of weightbetween 10² and 10⁶ Da.

The coating of the device may comprise alternate layers of a cationicpolymer and an anionic polymer. The cationic polymer may be a straightchain polymer but is more usually a branched chain polymer, ahyperbranched polymer or a polymer comprising a plurality of (cationic)hyperbranched polymer molecules, wherein, in the outer coating layer,there are covalently attached to said hyperbranched polymer moleculesone or more anti-coagulant entities via their functional end groups.

Thus, in one embodiment of the invention, one or more layers of thecoating, other than the outer layer, may be formed from the same orsimilar hyperbranched polymer molecules as the outer layer. Features ofsuch sub-layers may be as described for the outer layer, see Example 2.2and 3.3.

The device may comprise or be formed of a metal or a synthetic ornaturally occurring organic or inorganic polymer or a ceramic material,inter alia.

Thus, for example, it may be formed from a synthetic or naturallyoccurring organic or inorganic polymer or material such as polyethylene,polypropylene, polyacrylate, polycarbonate, polysaccharide, polyamide,polyurethane (PU), polyvinylchloride (PVC), polyetheretherketone (PEEK),cellulose, silicone or rubber (polyisoprene), plastics materials,metals, glass, ceramics and other known medical materials or acombination of such materials. Other suitable substrate materialsinclude fluoropolymers, e.g expanded polytetrafluoroethylene (ePTFE),polytetrafluoroethylene (PTFE), fluorinated ethylene-propylene (FEP),perfluorocarbon copolymers, e.g. tetrafluoroethylene perfluoroalkylvinylether (TFE/PAVE) copolymers, copolymers of tetrafluoroethylene (TFE) andperfluoromethyl vinyl ether (PMVE), and combinations of the above withand without crosslinking between the polymer chains.

Suitable metals include nickel titanium alloy (Nitinol), stainlesssteel, titanium, cobalt chromium, gold and platinum. Nitinol andstainless steel are preferred. Titanium is also preferred.

More generally, suitable metals include metallic materials and alloyssuch as cobalt chromium alloy (ELGILOY), stainless steel (316L), highnitrogen stainless steel, cobalt chrome alloy L-605, MP35N, MP2ON ,tantalum, nickel-titanium alloy, nitinol, platinum-iridium alloy, gold,magnesium, and combinations thereof.

We prefer the coated surface to which the anti-coagulant entity (e.g.heparin or other heparin moiety) is attached to be such that it retainsnon-thrombogenic properties after sterilization, e.g. ethylene oxide(EO) sterilization.

Sterilization may be carried out by means well known to those skilled inthe art. The preferred method of sterilization is using ethylene oxidegas. Alternatively, other methods such as radiation, e.g. e-beam orgamma radiation, may be used where such radiation will not degrade theobject or the coating or both.

A preferred embodiment of the present invention relates to a coatedmedical device for implantation e.g. permanent implantation, or otherplacement, at an anatomical site. Other preferred embodiments includetemporary use devices such as catheters and extracorporeal circuits.Examples are sterile (e.g. sterilized) medical devices for placementinside an anatomical structure delimiting a void space, or lumen, toreinforce the anatomical structure or maintain the void space. Suitablythe attached anti-coagulant entity, e.g. heparin or other heparinmoiety, does not elute to any substantial extent and remains with thedevice. For example, after 15 hour rinse with NaCl (0.15 M) prior totesting the retained AT binding capacity remains adequate (e.g. greaterthan 1 or 2 or 4 or 5 or 10 pmol/cm²) and/or when tested in the Bloodloop evaluation test (see Example 6) with fresh blood from a healthydonor the reduction in platelet count of the blood after the test issubstantially lower for the blood exposed to the coated surfaceaccording to the invention than that of an uncoated control (e.g. thereduction in platelet count after the test for the blood exposed to thecoated surface is less than 20%, preferably less than 15% and morepreferably less than 10%).

The non-thrombogenic character of devices according to the presentinvention may be tested by a number of methods. For examplenon-thrombogenic character may be associated with having a highantithrombin binding capacity, especially as compared with deviceshaving untreated surfaces.

For example, we prefer the surface of the device e.g. the medical deviceto have an antithrombin (AT) binding capacity of at least 1 (e.g. atleast 5) picomoles AT per square centimeter (pmol/cm²) of surface. Inother embodiments, the AT binding capacity is at least 6 pmol/cm², atleast 7 pmol/cm², at least 8 pmol/cm², at least 9 pmol/cm², or at least10 pmol/cm² of surface. In some embodiments, the AT binding capacity isat least 100 pmol/cm² of surface. AT binding capacity can be measured bymethods known in the art, e.g. those described in Pasche., et al., in“Binding of antithrombin to immobilized heparin under varying flowconditions” Artif. Organs 15:481-491 (1991) and US 2007/0264308. By wayof comparison it may be concluded from Sanchez et al (1997) J. Biomed.Mater. Res. 37(1) 37-42, see FIG. 1, that AT binding values of around2.7-4.8 pmol/cm² (depending on the experimental set up) or more do notappear to give rise to significant thrombogenic enzymatic activity uponcontact with plasma.

Alternatively or additionally we prefer the surface to benon-thrombogenic due to high capacity to suppress coagulation and otherdefence systems as shown in the Blood loop evaluation test described inExample 6. According to that test, the surface to be investigated isapplied to a PVC tubing which is rinsed for 15 hours with 0.15 M NaClprior to testing with fresh blood.

The thrombogenicity of an uncoated control surface is indicated by areduction in platelet count of the exposed blood, measured after thetest. The non-thrombogenicity of a surface prepared according to themethod described herein is indicated by a reduction in the plateletcount of the blood to a substantially lower degree (e.g. the reductionin platelet count after the test for the blood exposed to the coatedsurface is less than 20%, preferably less than 15% and more preferablyless than 10%).

Other similar blood evaluation methods different from the Blood loopmodel can be performed by those skilled in the art in order to assessthrombogenicity/non-thrombogenicity.

The amount of the anti-coagulant entity bound to a particular surfacearea can easily be controlled and adjusted by choosing particular sizesand amounts of hyperbranched molecule for the coating.

The distribution of the anti-coagulant entity on the surface can bedetermined by conventional staining techniques which are known per se,e.g. the distribution of heparin can be determined using toluidine blue.

Beneficial Agents Within the Coating

The layered coating of the device, particularly a medical device, maycomprise one or more beneficial agents besides the anti-coagulantentities. Exemplary beneficial agents include drug molecules andlubricious agents. The beneficial agent may be introduced to theunderlayers or to the outer coating layer.

Beneficial agents may be attached to the coating by a covalent linkage,which may be degradable to allow migration (i.e. elution) of thebeneficial agent from the polymer surface or it may not be degradable iflong lasting action is required. Alternatively, they may be adsorbedonto or incorporated within the coating surface (e.g. within any of itslayers) without covalent linkage.

In medical devices, it may be appropriate to attach drug molecules to ahyperbranched polymer of the layered coating (e.g. a hyperbranchedpolymer of the outer coating layer) in addition to the anti-coagulantentity. In one embodiment, the linkage between the drug molecules andthe coating is a degradable covalent linkage to allow migration (i.e.elution) of the drug molecules from the polymer surface. Alternatively,the drug may be adsorbed onto or incorporated within the coating surfacewithout covalent linkage. The drugs may also be incorporated into thevoids of the hyperbranched polymer prior to usage in the coating buildup. Hydrophobic drugs may, in particular, be incorporated into thehydrophobic voids of the hyperbranched polymer. A specific applicationof this is in drug eluting stents. Exemplary drugs that may be used inthis embodiment include drugs that prevent restenosis such asanti-angiogenic or anti-proliferative drugs such as paclitaxel andsirolimus. Another application is the use of elutable heparin or otheranti-coagulant entities. In another embodiment an antimicrobial drug maybe attached to the coating in addition to the anti-coagulant entity.

When beneficial agents are covalently attached to a molecule of thecoating, this may be achieved by covalently attaching beneficialagent(s) to cationic hyperbranched polymer molecules as described hereinthrough functional end groups which are not involved in attachment tothe anti-coagulant entity. These functional end groups may be theoriginal functionality (e.g. primary amine) or the functionality may bechanged prior to attachment to the beneficial agent. The coupling ofbeneficial agents may be conducted in a similar manner, as earlierdescribed, as for the coupling of anti-coagulant entities.

Beneficial agents may be coupled to hyperbranched polymers of theinvention before coupling of anti-coagulant entities, however moreusually they will be coupled afterwards.

More generally, the layered coating of the device (e.g. a medicaldevice) may optionally comprise at least one beneficial agent selectedfrom: paclitaxel, a taxane or other paclitaxel analogue; estrogen orestrogen derivatives; heparin or another thrombin inhibitor, hirudin,hirulog, apyrase, argatroban, D-phenylalanyl-L-poly-L-arginylchloromethyl ketone, or another antithrombogenic agent, or mixturesthereof; urokinase, streptokinase, a tissue plasminogen activator, oranother thrombolytic agent, or mixtures thereof; a fibrinolytic agent; avasospasm inhibitor; a calcium channel blocker, a nitrate, nitric oxide,a nitric oxide promoter or another vasodilator; aspirin, ticlopidine oranother antiplatelet agent; vascular endothelial growth factor (VEGF) oranalogues thereof; colchicine or another antimitotic, or anothermicrotubule inhibitor; cytochalasin or another actin inhibitor; aremodeling inhibitor; deoxyribonucleic acid, an antisense nucleotide oranother agent for molecular genetic intervention; a cell cycle inhibitor(such as the protein product of the retinoblastoma tumor suppressorgene), or analogues thereof GP IIb/IIIa, GP Ib-IX or another inhibitoror surface glycoprotein receptor; methotrexate or another antimetaboliteor antiproliferative agent; an anti-cancer chemotherapeutic agent;dexamethasone, dexamethasone sodium phosphate, dexamethasone acetate oranother dexamethasone derivative, or another anti-inflammatory steroid;prostaglandin, prostacyclin or analogues thereof; an immunosuppressiveagent (such as cyclosporine or rapamycin (also known as sirolimus) andanalogues thereof); an antimicrobial agent (e.g. compounds selected fromthe group consisting of diamidines, iodine and iodophors, peroxygens,phenols, bisphenols, halophenols, biguanides, silver compounds,triclosan, chlorhexidine, triclocarban, hexachlorophene,dibromopropamidine, chloroxylenol, phenol and cresol or combinationsthereof) an antibiotic, erythromycin orvancomycin; dopamine,bromocriptine mesylate, pergolide mesylate or another dopamine agonist;or another radiotherapeutic agent; iodine-containing compounds,barium-containing compounds, gold, tantalum, platinum, tungsten oranother heavy metal functioning as a radiopaque agent; a peptide, aprotein, an enzyme, an extracellular matrix component, a cellularcomponent or another biologic agent; captopril, enalapril or anotherangiotensin converting enzyme (ACE) inhibitor; ascorbic acid,alphatocopherol, superoxide dismutase, deferoxyamine, a 21-aminosteroid(lasaroid) or another free radical scavenger, iron chelator orantioxidant; angiopeptin; a ¹⁴C—, ³H—, ¹³¹I—, ³²P or ³⁶S-radiolabelledform or other radiolabelled form of any of the foregoing; or a mixtureof any of these.

Further beneficial agents that may be incorporated into the surface ofthe device include lubricious agents including polymers such ashydrophilic or hydrogel polymers containing polar or charged functionalgroups which render them soluble in water. These agents incorporatepolar groups that have an affinity to water molecules in solution, andare broadly classified as hydrogels. It may also be appropriate toattach lubricious agents to the coating in addition to theanti-coagulant entity. In one embodiment, the linkage between thelubricious agents and the outer coating layer may be a covalent linkage.Alternatively, the lubricious agent may be adsorbed ionically orphysically onto or incorporated within the coating surface withoutcovalent linkage. Examples of lubricous agents are, but are not limitedto, hyaluronic acid, hyaluronic acid derivatives,poly-N-vinylpyrrolidone, poly-N-vinylpyrrolidone derivatives,polyethylene oxide, polyethylene oxide derivatives, polyethylene glycol,polyethylene glycol derivatives, polyvinylalcohol, polyvinylalcoholderivatives, polyacrylic acid, polyacrylic acid derivatives, silicon,silicon derivatives, polysaccharide, polysaccharide derivatives,Sulfonated polystyrene, Sulfonated polystyrene derivatives,polyallylamine, polyallylamine derivatives, polyethyleneimine,polyethyleneimine derivatives, polyoxazoline, polyoxazoline derivatives,polyamine, polyamine derivatives and combinations thereof. Suchbeneficial agents may, for example, be covalently attached tohyperbranched polymer molecules in the outer coating layer.

When a device has several surfaces, the beneficial agent(s) may beincorporated into which ever surface is appropriate to achieve thebeneficial effect. For example the beneficial agent(s) may beincorporated into the surface of a tubular device on either or both ofthe luminal and abluminal sides. When more than one beneficial agent isincorporated, the different beneficial agents may be incorporated intothe same surface, or part of the surface, or different surfaces or partsof the surface.

The devices of the invention may have one or more of the followingadvantages in at least some embodiments:

-   -   The amount of the entity coupled to the outer most layer may be        controlled;    -   Both end-point (one point) attachment and multi-point attachment        of the entity, e.g. heparin, can be achieved, although end point        (especially reducing end point) attachment is preferred;    -   The length of the covalent connection (linker(s) and spacer(s))        between the entity and the hyperbranched polymer may be        controlled;    -   Full length heparin may be used thus avoiding the cleavage of        heparin and thus optimizing the use of heparin raw material;    -   Use of full-length heparin or heparin linked via a spacer may        improve the bioactivity of the bound heparin;    -   A uniform distribution of the entity over the outer coating        layer can be obtained;    -   A uniform coating may be obtained which will mask the intrinsic        properties, for example lower the thromogenic properties, of a        device irrespective of the material of its manufacture;    -   A coating may be obtained which is comparatively smooth and/or        lubricious;    -   The bioavailability of the anti-coagulant entity can be        controlled and improved;    -   A non-thrombogenic coating which does not leach heparin and        therefore has long lifetime may be obtained;    -   A coating whose properties are preserved upon aging may be        obtained;    -   A coating whose properties are preserved upon sterilization        (e.g. with EO) may be obtained;    -   A self-healing coating may be obtained due to the possibility of        reversible forming of ionic interactions between the layers;    -   The number of steps for coating preparation may be minimised by        using pre-fabricated components;    -   A robust manufacturing process can be obtained by using        pre-fabricated components;    -   A coating may be prepared in which a pre-prepared conjugate with        covalently bound heparin may be used in the coating build up        process;    -   The biocompatibility of the prepared coating may be enhanced;    -   A coating according to the present invention may reduce the need        for systemic heparin, and reduce the likelihood of contact        activation;    -   A medical device having a combination of lubricity and        thromboresistance can be obtained which may be beneficial in        certain applications e.g. neuro vascular applications;    -   A medical device having a combination of drug eluting properties        and thromboresistance can be obtained which may be beneficial in        certain applications e.g. drug eluting stents and drug eluting        balloons;    -   A medical device having a combination of anti-inflammatory        properties and thromboresistance can be obtained which may be        beneficial in certain applications e.g. cardiovascular        applications;    -   An analytical or separation device with improved binding        capacity to biomolecules may be obtained; and    -   An analytical or separation device with extended heparin        activity life time may be obtained.

The invention is illustrated, but in no way limited, by the followingExamples:

EXAMPLES

All Lupasol samples were purchased from BASF. Lupasol® WF (ethylenediamine core) has an average molecular weight of 25 kDa as determinedfrom light scattering. Dextrane sulfate was purchased from pK ChemicalsA/S (PKC) and PAMAM dendrimers (ethylene diamine core) were purchasedfrom Sigma Aldrich and Dendritech. PAMAM-G6.0-NH₂ is a PAMAM dendrimer(6^(th) generation) with molecular weight of approximately 60 kDa.PAMAM-G8.0-NH₂ is a PAMAM dendrimer (8^(th) generation) with molecularweight of approximately 230 kDa. PPI G5 dendrimer (butane-1, 4-diaminecore) was purchased from Aldrich. PPI G5 is a dendrimer (5^(th)generation) with a molecular weight of approximately 7 kDa. Thepolyamine Epomin P-1050 (ethylene diamine core) was purchased fromNippon Shokubai and has an average molecular weight of 70 kDa. Thepolyamine G-35 was purchased from Wuhan Bright Chemicals and has anaverage molecular weight of 70 kDa. All polyamine stock solutions were5wt % in water. The dextran sulfate stock solution was 6wt % in water.The solutions were subsequently diluted as appropriate before use. Awater rinse was performed in between each process step as appropriate.

Example Headings

-   -   1. Preparation of underlayer    -   2. Preparation of a non-thrombogenic coating comprising a        hyperbranched polymer in the outer coating layer    -   3. Preparation of a non-thrombogenic coating comprising a        pre-prepared heparin functionalized hyperbranched polymer in the        outer coating layer    -   4. Derivatized heparin entities    -   5. Derivatized hyperbranched polymers    -   6. Evaluation of heparin density and blood platelet loss    -   7. Preparation of intermediates    -   8. Preparation of a hydrophilic and lubricious coatings    -   9. Preparation of drug eluting coatings    -   10. Biocompatibility study    -   11. Hemo-compatibility of EO sterilized coatings comprising        hyperbranched polymers

Example 1 Preparation of Underlayer Example 1.1 Preparation ofUnderlayer Comprising Lupasol® SN

A PVC surface was pretreated using the method described by Larm et al inEP-B-0086186 and EP-495820 (layer-by-layer; polyelectrolyte chargeinteractions) ending with a layer of sulfated polysaccharide.

The luminal surface of a PVC tubing (I.D. 3 mm) was cleaned withisopropanol and an oxidizing agent. The priming was built-up byalternated adsorption of a positively charged polyamine (Lupasol® SN, 5wt % in water) and negatively charged sulfated polysaccharide (dextransulfate, 6 wt % in water). The polyamine was crosslinked with adifunctional aldehyde (crotonaldehyde). Every pair of polyamine andsulfated polysaccharide is called one bilayer. The PVC surface wasprimed with 3 bilayers ending with the sulfated polysaccharide.

Example 1.2 Preparation of Underlayer Comprising Lupasol® WF

A PVC surface was pretreated using the method described by Larm et al inEP-B-0086186 and EP-495820 (layer-by-layer; polyelectrolyte chargeinteractions) ending with a layer of sulfated polysaccharide.

The luminal surface of a PVC tubing (I.D. 3 mm) was cleaned withisopropanol and an oxidizing agent. The priming was built-up byalternated adsorption of a positively charged polyamine (Lupasol® WF, 5wt % in water) and negatively charged sulfated polysaccharide (dextransulfate, 6 wt % in water). The polyamine was crosslinked with adifunctional aldehyde (crotonaldehyde). Every pair of polyamine andsulfated polysaccharide is called one bilayer. The PVC surface wasprimed with 3 bilayers ending with the sulfated polysaccharide.

Example 1.3 Preparation of Underlayer Comprising PAMAM-G6.0-NH₂Dendrimer

Quartz Crystal Microbalance (QCM) crystals covered with gold (QSX 301,Q-Sense) were coated according to Example 1.1 using 5wt % in MeOHPAMAM-G6.0-NH₂ (1 mL/L) to obtain a 3 bilayer coating consisting ofalternatively layers of PAMAM-G6.0-NH₂ and a sulfated polysaccharide (6wt % in water). The polyamine was crosslinked with a difunctionalaldehyde (crotonaldehyde). A 2 min water rinse is conducted in betweeneach adsorption step. The gold surface was primed with 3 bilayers endingwith the sulfated polysaccharide.

Example 1.4 Preparation of Underlayer Comprising Lupasol® SK and HeparinFunctionalized PAMAM-G6.0-NH₂ Dendrimer

The luminal surface of a PVC tubing (I.D. 3 mm) was cleaned withisopropanol and an oxidizing agent. The priming was built-up byalternated adsorption of a positively charged polyamine (Lupasol® SK, 5wt % in water, 10 minutes) and negatively charged PAMAM-heparinconjugate (400 mg/L, from Example 5.2, 20 minutes). The polyamine wascrosslinked with a difunctional aldehyde (crotonaldehyde). Every pair ofpolyamine and PAMAM-heparin conjugate is called one bilayer. The PVCsurface was primed with 3 bilayers ending with the PAMAM-heparinconjugate from Example 5.2.

Example 1.5 Preparation of Underlayer Using Lupasol® WF and HeparinFunctionalized PAMAM-G6.0-NH₂ Dendrimer

The luminal surface of a PVC tubing (I.D. 3 mm) was cleaned withisopropanol and an oxidizing agent. The priming was built-up byalternated adsorption of a positively charged polyamine (Lupasol® WF, 5wt % in water, 10 minutes) and negatively charged PAMAM-heparinconjugate (400 mg/L, from Example 5.2, 20 minutes). The polyamine wascrosslinked with a difunctional aldehyde (crotonaldehyde). Every pair ofpolyamine and PAMAM-heparin conjugate is called one bilayer. The PVCsurface was primed with 3 bilayers ending with the PAMAM-heparinconjugate from Example 5.2.

Example 1.6 Preparation of Underlayer Comprising G-35

The luminal surface of a PVC tubing (I.D. 3 mm) was cleaned withisopropanol and an oxidizing agent. The priming was built-up byalternated adsorption of a positively charged polyamine (G-35, 5 wt % inwater) and negatively charged sulfated polysaccharide (dextran sulfate,6 wt % in water). The polyamine is crosslinked with a difunctionalaldehyde (crotonaldehyde). Every pair of polyamine and sulfatedpolysaccharide is called one bilayer. The PVC surface was primed with 3bilayers ending with the sulfated polysaccharide.

Example 1.7 Preparation of Underlying Layers Using Lupasol® SK

The luminal surface of a PVC tubing (I.D. 3 mm) was cleaned withisopropanol and an oxidizing agent. The priming was built-up byalternated adsorption of a positively charged polyamine (Lupasol® SK, 5wt % in water) and negatively charged sulfated polysaccharide (dextransulfate, 6 wt % in water). The polyamine was crosslinked with adifunctional aldehyde (crotonaldehyde).

Every pair of polyamine and sulfated polysaccharide is called onebilayer. The PVC surface was primed with 3 bilayers ending with thesulfated polysaccharide.

Example 1.8 Preparation of Underlying Layers Using Epomin P-1050

The luminal surface of a PVC tubing (I.D. 3 mm) was cleaned withisopropanol and an oxidizing agent. The priming was built-up byalternated adsorption of a positively charged polyamine (Epomin P-1050,5 wt % in water) and negatively charged sulfated polysaccharide (dextransulfate, 6 wt % in water). The polyamine was crosslinked with adifunctional aldehyde (crotonaldehyde). Every pair of polyamine andsulfated polysaccharide is called one bilayer. The PVC surface wasprimed with 3 bilayers ending with the sulfated polysaccharide.

Example 2 Preparation of a non-Thrombogenic Coating Comprising aHyperbranched Polymer in the Outer Coating Layer Example 2.1 Preparationof Outer Coating Layer Comprising Lupasol® WF On Underlayer ComprisingLupasol® SN

A solution of Lupasol® WF (5 wt %) was allowed to adsorb for 10 minutesto the prefabricated coating surface from Example 1.1 followed by a 1hour coupling step of nitrous acid degraded heparin (325 mg/L), fromExample 4.1, using a reducing agent (sodium cyanoborohydride, 2.5 wt %in water). A 2 min water rinse is conducted in between each adsorptionstep. The fabricated non-thrombogenic coating was treated with aborate/phosphate solution to remove any potential ionically boundheparin prior to evaluation of its non-thrombogenic properties.

Example 2.2 Preparation of Outer Coating Layer Comprising Lupasol® WF onUnderlayer Comprising Lupasol® WF

A solution of Lupasol® WF (5 wt %) was allowed to adsorb for 10 minutesto the prefabricated coating surface from Example 1.2 followed by a 1hour coupling step of nitrous acid degraded heparin (325 mg/L), fromExample 4.1, using a reducing agent (sodium cyanoborohydride, 2.5 wt %in water). A 2 min water rinse is conducted in between each adsorptionstep. The fabricated non-thrombogenic coating was treated with aborate/phosphate solution to remove any potential ionically boundheparin prior to evaluation of its non-thrombogenic properties.

Example 2.3 Preparation of Outer Coating Layer Comprising PAMAM-G6.0-NH₂Dendrimer on Underlayer Comprising Lupasol® SN

A solution of PAMAM-G6.0-NH₂ (5 wt %) was allowed to adsorb for 10minutes to the prefabricated coating surface from Example 1.1 followedby a 1 hour coupling step of nitrous acid degraded heparin (325 mg/L),from Example 4.1, using a reducing agent (sodium cyanoborohydride, 2.5wt % in water). A 2 min water rinse is conducted in between eachadsorption step. The fabricated non-thrombogenic coating was treatedwith a borate/phosphate solution to remove any potential ionically boundheparin prior to evaluation of its non-thrombogenic properties.

Example 2.4 Preparation of Outer Coating Layer Comprising PAMAM-G6.0-NH₂Dendrimer on Underlayer Comprising PAMAM-G6.0-NH₂ Dendrimer

A solution of PAMAM-G6.0-NH₂ (5 wt %) was allowed to adsorb for 30minutes to the prefabricated coating surface from Example 1.3 followedby a 1 hour coupling step of nitrous acid degraded heparin (325 mg/L),from Example 4.1, using a reducing agent (sodium cyanoborohydride, 2.5wt % in water). A 2 min water rinse is conducted in between eachadsorption step. The fabricated non-thrombogenic coating was treatedwith a borate/phosphate solution to remove any potential ionically boundheparin prior to evaluation of its non-thrombogenic properties.

Example 2.5 Preparation of Outer Coating Layer Comprising G-35 onUnderlayer Comprising Lupasol® SN

A solution of G-35 (5 wt %) was allowed to adsorb for 10 minutes to theprefabricated coating surface from Example 1.1 followed by a 1 hourcoupling step of nitrous acid degraded heparin (325 mg/L), from Example4.1, using a reducing agent (sodium cyanoborohydride, 2.5 wt % inwater). A 2 min water rinse is conducted in between each adsorptionstep. The fabricated non-thrombogenic coating was treated with aborate/phosphate solution to remove any potential ionically boundheparin prior to evaluation of its non-thrombogenic properties.

Example 2.6 Preparation of Outer Coating Layer Comprising G-35 onUnderlayer Comprising G-35

A solution of G-35 (5 wt %) was allowed to adsorb for 10 minutes to theprefabricated coating surface from Example 1.6 followed by a 1 hourcoupling step of nitrous acid degraded heparin (325 mg/L), from Example4.1, using a reducing agent (sodium cyanoborohydride, 2.5 wt % inwater). A 2 min water rinse is conducted in between each adsorptionstep. The fabricated non-thrombogenic coating was treated with aborate/phosphate solution to remove any potential ionically boundheparin prior to evaluation of its non-thrombogenic properties.

Example 2.7 Preparation of Outer Coating Layer Comprising 10 wt %Lupasol® WF and 90 wt % Lupasol® SN on Underlayer Comprising Lupasol® SN

A mixture of 10 wt % Lupasol® WF (5 wt % solution) and 90 wt % Lupasol®SN (5 wt % solution) was allowed to adsorb for 10 minutes to theprefabricated surface from Example 1.1 followed by a 1 hour couplingstep of nitrous acid degraded heparin (325 mg/L), from Example 4.1,using a reducing agent (sodium cyanoborohydride, 2.5 wt % in water). A 2min water rinse is conducted in between each adsorption step. Thefabricated non-thrombogenic coating was treated with a borate/phosphatesolution to remove any potential ionically bound heparin prior toevaluation of its non-thrombogenic properties.

Example 2.8 Preparation of Outer Coating Layer Comprising 10 wt %Lupasol® WF and 90 wt % Lupasol® SK on Underlayer Comprising Lupasol® SK

A mixture of 10 wt % Lupasol® WF (5 wt % solution) and 90 wt % Lupasol®SK (5 wt % solution) was allowed to adsorb for 10 minutes to theprefabricated coating surface from Example 1.7 followed by a 1 hourcoupling step of nitrous acid degraded heparin (325 mg/L), from Example4.1, using a reducing agent (sodium cyanoborohydride, 2.5 wt % inwater). A 2 min water rinse is conducted in between each adsorptionstep. The fabricated non-thrombogenic coating was treated with aborate/phosphate solution to remove any potential ionically boundheparin prior to evaluation of its non-thrombogenic properties.

Example 2.9 Preparation of Outer Coating Layer Comprising Lupasol® WF onUnderlayer Comprising Lupasol® SK

A solution of Lupasol® WF (5 wt %) was allowed to adsorb for 10 minutesto the prefabricated coating surface from Example 1.7 followed by a 1hour coupling step of nitrous acid degraded heparin (325 mg/L), fromExample 4.1, using a reducing agent (sodium cyanoborohydride, 2.5 wt %in water). A 2 min water rinse is conducted in between each adsorptionstep. The fabricated non-thrombogenic coating was treated with aborate/phosphate solution to remove any potential ionically boundheparin prior to evaluation of its non-thrombogenic properties.

Example 2.10 Preparation of Outer Coating Layer Comprising Epomin P-1050on Underlayer Comprising Lupasol® SN

A solution of Epomin P-1050 (5 wt %) was allowed to adsorb for 10minutes to the prefabricated coating surface from Example 1.1 followedby a 1 hour coupling step of nitrous acid degraded heparin (325 mg/L),from Example 4.1, using a reducing agent (sodium cyanoborohydride, 2.5wt % in water). A 2 min water rinse is conducted in between eachadsorption step. The fabricated non-thrombogenic coating was treatedwith a borate/phosphate solution to remove any potential ionically boundheparin prior to evaluation of its non-thrombogenic properties.

Example 2.11 Preparation of Outer Coating Layer Comprising HeparinizedLupasol® WF on Underlayer Comprising Lupasol® SN

Lupasol® WF (5 wt % in water) was allowed to adsorb onto an underlayinglayer described essentially as in Example 1.1 yielding a positivelycharged surface. Na heparin (325 mg/L) was subsequently coupled to thepositively charged layer using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (23.35 mg/L) at room temperature for 60 minutesfollowed by a borate/phosphate rinse to remove any loosely bound heparinprior to evaluation of the non-thrombogenic effect of the coating.

Example 2.12 Preparation of Outer Coating Layer Comprising ApyraseFunctionalized Lupasol® WF on Underlayer Comprising Lupasol® SN

Apyrase, ≥200 units/mg protein, derived from potato was purchased fromSigma-Aldrich. The carboxylic content in apyrase was calculated to beapproximately 90 moles of COOH per 1 mole of apyrase based on an aminoacid analysis performed by Aminosyraanalyscentralen, Sweden. Carboxylicgroups in non-thrombogenic agents, such as apyrase, may be used to linkthem to an amine containing hyperbranched polymer using EDC, or EDClike, reagents essentially as described in Example 2.11.

Example 3 Preparation of a Non-Thrombogenic Coating Comprising aPre-Prepared Heparin Functionalized Hyperbranched Polymer in the OuterCoating Layer Example 3.1 Preparation of Outer Coating Layer ComprisingHeparin Functionalized PAMAM-G6.0-NH₂ Dendrimer on Underlayer ComprisingLupasol® SN

The luminal surface of a PVC tubing (I.D. 3 mm) was cleaned withisopropanol and an oxidizing agent. The priming was built-up byalternated adsorption of a positively charged polyamine (Lupasol® SN, 5wt % in water) and negatively charged sulfated polysaccharide (dextransulfate, 6 wt % in water). The polyamine was crosslinked with adifunctional aldehyde (crotonaldehyde). Every pair of polyamine andsulfated polysaccharide is called one bilayer. The PVC surface wasprimed with 3 bilayers and one layer of Lupasol® SN. Heparinfunctionalized PAMAM-G6.0-NH₂ dendrimer (150 mg/L) from Example 5.2 wasdeposited onto the positively charged Lupasol® SN coating for 1 hourfollowed by a borate/phosphate rinse to remove any loosely bound heparinconjugate prior to evaluation of the non-thrombogenic effect of thecoating.

Example 3.2 Preparation of Outer Coating Layer Comprising HeparinFunctionalized PAMAM-G6.0-NH₂ Dendrimer on Underlayer ComprisingLupasol® SK, Lupasol® WF and Heparin Functionalized PAMAM-G6.0-NH₂Dendrimer

The luminal surface of a PVC tubing (I.D. 3 mm) was cleaned withisopropanol and an oxidizing agent. The priming was built-up byalternated adsorption of a positively charged polyamine (Lupasol® SK, 5wt % in water) and negatively charged PAMAM-heparin conjugate (400 mg/L,from Example 5.2). The polyamine was crosslinked with a difunctionalaldehyde (crotonaldehyde). Every pair of polyamine and PAMAM-heparinconjugate is called one bilayer. The PVC surface was primed with 3bilayers as just described (also, see Example 1.4) followed by one layerof Lupasol® WF. Heparin functionalized PAMAM-G6.0-NH₂ dendrimer (400mg/L) from Example 5.2 was deposited onto the positively chargedLupasol® WF coating for 20 minutes followed by a water rinse to removeany loosely bound heparin conjugate prior to evaluation of thenon-thrombogenic effect of the coating.

Example 3.3 Preparation of Outer Coating Layer Comprising HeparinFunctionalized PAMAM-G6.0-NH₂ Dendrimer on Underlayer ComprisingLupasol® WF and Heparin Functionalized PAMAM-G6.0-NH₂ Dendrimer

The luminal surface of a PVC tubing (I.D. 3 mm) was cleaned withisopropanol and an oxidizing agent. The priming was built-up byalternated adsorption of a positively charged polyamine (Lupasol® WF, 5wt % in water) and negatively charged PAMAM-heparin conjugate (400 mg/L,from Example 5.2). The polyamine was crosslinked with a difunctionalaldehyde (crotonaldehyde). Every pair of polyamine and PAMAM-heparinconjugate is called one bilayer. The PVC surface was primed with 3bilayers as just described (also, see Example 1.5) followed by one layerof Lupasol® WF. Heparin functionalized PAMAM-G6.0-NH₂ dendrimer (400mg/L) from Example 5.2 was deposited onto the positively chargedLupasol® WF coating for 20 minutes followed by a water rinse to removeany loosely bound heparin conjugate prior to evaluation of thenon-thrombogenic effect of the coating.

Example 3.4 Preparation of Outer Coating Layer Comprising HeparinFunctionalized PAMAM-G6.0-NH₂ Dendrimer on Underlayer ComprisingLupasol® SN

The luminal surface of a PVC tubing (I.D. 3 mm) was cleaned withisopropanol and an oxidizing agent. The priming was built-up byalternated adsorption of a positively charged polyamine (Lupasol® SN, 5wt % in water) and negatively charged sulfated polysaccharide (dextransulfate, 6 wt % in water). The polyamine was crosslinked with adifunctional aldehyde (crotonaldehyde). Every pair of polyamine andsulfated polysaccharide is called one bilayer. The PVC surface wasprimed with 3 bilayers and one layer of Lupasol® SN. Heparinfunctionalized PAMAM-G6.0-NH₂ dendrimer (425 mg/L) from Example 5.2 wasdeposited onto the positively charged Lupasol® SN coating for 1 hourfollowed by a borate/phosphate rinse to remove any loosely bound heparinconjugate prior to evaluation of the non-thrombogenic effect of thecoating.

Example 3.5 Preparation of Outer Coating Layer Comprising HeparinFunctionalized Lupasol® WF on Underlayer Comprising Lupasol® SN

The luminal surface of a PVC tubing (I.D. 3 mm) was cleaned withisopropanol and an oxidizing agent. The priming was built-up byalternated adsorption of a positively charged polyamine (Lupasol® SN, 5wt % in water) and negatively charged sulfated polysaccharide (dextransulfate, 6 wt % in water). The polyamine was crosslinked with adifunctional aldehyde (crotonaldehyde). Every pair of polyamine andsulfated polysaccharide is called one bilayer. The PVC surface wasprimed with 3 bilayers and one layer of Lupasol® SN. Heparinfunctionalized Lupasol® WF (425 mg/L) from Example 5.3 was depositedonto the positively charged Lupasol® SN coating for 1 hour followed by aborate/phosphate rinse to remove any loosely bound heparin conjugateprior to evaluation of the non-thrombogenic effect of the coating.

Example 3.6 Preparation of Outer Coating Layer Comprising HeparinFunctionalized PAMAM-G8.0-NH₂ Dendrimer on Underlayer ComprisingLupasol® SN

The luminal surface of a PVC tubing (I.D. 3 mm) was cleaned withisopropanol and an oxidizing agent. The priming was built-up byalternated adsorption of a positively charged polyamine (Lupasol® SN, 5wt % in water) and negatively charged sulfated polysaccharide (dextransulfate, 6 wt % in water). The polyamine was crosslinked with adifunctional aldehyde (crotonaldehyde). Every pair of polyamine andsulfated polysaccharide is called one bilayer. The PVC surface wasprimed with 3 bilayers and one layer of Lupasol® SN. Heparinfunctionalized PAMAM-G8.0-NH₂ dendrimer (425 mg/L) from Example 5.6 wasdeposited onto the positively charged Lupasol® SN coating for 1 hourfollowed by a borate/phosphate rinse to remove any loosely bound heparinconjugate prior to evaluation of the non-thrombogenic effect of thecoating.

Example 3.7 Preparation of Outer Coating Layer Comprising HeparinFunctionalized PPI G5 Dendrimer on Underlayer Comprising Lupasol® SN

The luminal surface of a PVC tubing (I.D. 3 mm) was cleaned withisopropanol and an oxidizing agent. The priming was built-up byalternated adsorption of a positively charged polyamine (Lupasol® SN, 5wt % in water) and negatively charged sulfated polysaccharide (dextransulfate, 6 wt % in water). The polyamine was crosslinked with adifunctional aldehyde (crotonaldehyde). Every pair of polyamine andsulfated polysaccharide is called one bilayer. The PVC surface wasprimed with 3 bilayers and one layer of Lupasol® SN. Heparinfunctionalized PPI G5 dendrimer (425 mg/L) from Example 5.7 wasdeposited onto the positively charged Lupasol® SN coating for 1 hourfollowed by a borate/phosphate rinse to remove any loosely bound heparinconjugate prior to evaluation of the non-thrombogenic effect of thecoating.

Example 4 Derivatized Heparin Entities Example 4.1 Preparation ofAldehyde End-Point Functionalized Heparin

Aldehyde functionalized heparin is prepared essentially as in Example 2of U.S. Pat. No. 4,613,665.

Example 4.2 Preparation of Thiol End-Point Functionalized Heparin

Nitrous acid degraded heparin with aldehyde groups (prepared essentiallyas in Example 2 of U.S. Pat. No. 4,613,665) (5.00 g, 1.0 mmol),cysteamine hydrochloride (0.57 g, 5.0 mmol) and sodium chloride (0.6 g)were dissolved in purified water. The pH was adjusted to 6.0 with 1 MNaOH (aq) and 1 M HCl (aq). To the solution was added 3.1 ml of 5% (aq)NaCNBH₃ (0.16 g, 2.5 mmol) and the reaction was stirred over night atroom temperature. The pH was adjusted to 11.0 with 1 M NaOH (aq) and theresulting product was dialyzed against purified water with a SpectraPordialysis membrane (MWCO 1 kD, flat width 45 mm) for three days. Thereaction mixture was then concentrated and freeze dried to obtain 2.6 gof a white fluffy powder.

Example 4.3 Preparation of Alkyne End-Point Functionalized Heparin

Alkyne functionalized nitrous acid degraded heparin is preparedessentially as in Example 3a of WO2010/029189.

Example 4.4 Preparation of Alkyne End-Point Functionalized NativeHeparin

Alkyne functionalized native heparin prepared essentially as in Example3b of WO2010/029189.

Example 4.5 Preparation of Azide End-Point Functionalized Heparin andAzide Functionalized Native Heparin

Azide functionalized nitrous acid degraded heparin and azidefunctionalized native heparin is prepared essentially as in Example 4 ofWO2010/029189.

Example 5 Derivatized Hyperbranched Polymers Example 5.1 Preparation ofAlkene Functionalized PAMAM-G6.0-NH₂ Dendrimer

A stock solution with 3.75 mg of NHS activated 5-hexenoic acid/mL MeOHwas prepared. See Example 7.1 for preparation of NHS activated alkene.

2 mL of a 5 wt % PAMAM-G6.0-NH₂ solution in MeOH was added to 1 mL ofthe stock solution (3.75 mg of NHS activated 5-hexenoic acid) and 9 mLof MeOH (0° C.). The reaction was allowed to proceed over night. Thesolvent was evaporated using a rotary evaporator and a vacuum oven. Highpurity of the obtained material was confirmed by ¹H and ¹³C NMR. Afunctionalization degree of 2% was obtained (5-6 alkenes/dendrimer)

Example 5.2 Preparation of Heparin Functionalized PAMAM-G6.0-NH₂Dendrimer With Preserved Specific Activity

Aldehyde end-point functionalized heparin, from Example 4.1, (5.0 g,0.56 mmol) was dissolved in 15 mL acetate buffer (pH=5.0) undervigorously stirring. 2 mL of a 5 wt % solution of PAMAM-G6.0-NH₂dendrimer (ethylene diamine core) (80.6 mg, 1.39 mol) in MeOH was addedto the heparin solution followed by addition of 10 mL sodiumcyanoborohydride (2.5 wt % in H₂O). The solution was left to stir in thefume hood over night at room temperature. The solution was transferredto a dialysis bag (MWCO 50,000 Da) and dialyzed thoroughly. The contentof the dialysis bag was thereafter transferred to a round bottom flaskand lyophilized over night. The dry weight of the content in the flaskwas 830 mg (˜60 heparin chains/PAMAM-G6.0-NH₂ dendrimer or 23%functionalization of the primary amines in PAMAM-G6.0-NH₂). The specificactivity of the PAMAM bound heparin in the conjugate was determined tobe >100 IU/mg. The heparin used for the preparation, prior to coupling,has a specific activity of approximately 100 IU/mg.

Example 5.3 Preparation of Heparin Functionalized Lupasol® WF withPreserved Specific Activity

Heparin functionalized Lupasol® WF was prepared essentially as describedin Example 5:2.

Example 5.4 Preparation of Azide Functionalized Lupasol® WF

Azide functionalized Lupasol® WF can be prepared essentially asdescribed for Lupasol® SN in Example 2a of WO2010/029189

Example 5.5 Preparation of Alkyne Functionalized Lupasol WF

Alkyne functionalized Lupasol® WF can be prepared essentially asdescribed for Lupasol® SN in Example 2b of WO2010/029189

Example 5.6 Preparation of Heparin Functionalized PAMAM-G8.0-NH₂Dendrimer With Preserved Specific Activity

Heparin functionalized PAMAM-G8.0-NH₂ was prepared essentially asdescribed in Example 5:2.

Example 5.7 Preparation of Heparin Functionalized PPI G5 Dendrimer WithPreserved Specific Activity

Heparin functionalized PPI G5 dendrimer was prepared essentially asdescribed in Example 5:2.

Example 5.8 Preparation of Functionalized Hyperbranched Polymers

Hyperbranched polymers with chemical groups, or functionalities,selected from Table 3 (Func. group 1 and Func. group 2) may be preparedby a person skilled in the art.

Non-thrombogenic entities (e.g. heparin) with chemical groups, orfunctionalities, selected from Table 3 (Func. 1 and Func. 2) may beprepared by a person skilled in the art.

The functionalized hyperbranched polymers may be reacted with afunctionalized non-thrombogenic entity (e.g. heparin) by a personskilled in the art to yield a hyperbranched polymer derivatised with anon-thromogenic entity (e.g. heparin).

Example 6 Evaluation of Heparin Density and Blood Platelet Loss

Heparin Density Test (For Measurement of the Heparin Content in theCoating)

Quantification of surface immobilized heparin was performed essentiallyas described in Smith R. L. and Gilkerson E (1979), Anal. Biochem., 98,478-480.

Toluidine Blue Staining Test (For Evaluation of Heparin Distribution)

Heparin distribution is evaluated using Toluidine blue stainingsolutions. The solution was prepared by dissolving 200 mg of Toluidineblue in 1 L of water. The samples were subjected to the stainingsolution for 2 minutes prior to extensive water rinse. A blue/violetstaining indicates that negatively charged heparin molecules arehomogenously distributed in the outer coating layer as exemplified byFIG. 9 plate B.

Blood Loop Evaluation Test (For Measurement of Platelet Loss)

Blood loop evaluation was performed on samples, as coated, to show thepreserved heparin bioactivity of the non-thrombogenic surface. First theluminal side of the coated tubing was washed with 0.15 M NaCl for 15hours at a flow of 1 mL/min to ensure that all loosely bound heparin wasrinsed off and a stable surface remains. Then the washed tubings wereincubated in a Chandler loop model performed essentially according toAndersson et al. (Andersson, J.; Sanchez, J.; Ekdahl, K. N.; Elgue, G.;Nilsson, B.; Larsson, R. J Biomed Mater Res A 2003, 67(2), 458-466) at20 rpm. The platelets, from fresh blood and from the blood collectedfrom the loops, were counted in a cell counter to measure the loss ofplatelets which indicates thrombosis.

Example 6.1 Coating Properties in Terms of Heparin Density and PlateletLoss, After Blood Exposure, of the Non-Thrombogenic Surface

Neg. charged Hyperbranched Heparin Toluidine Example Polyamine inpolymer in polymer in outer density^(a) blue Platelets No. underlayerunderlayer coating layer [μg/cm²] staining^(b) loss [%] 1.1 Lupasol ® SNPS* N/A ** N/A ** No N/A ** 1.2 Lupasol ® WF PS* N/A ** N/A ** No N/A **2.1 Lupasol ® SN PS* Lupasol ® WF 4.7 Yes 0 2.2 Lupasol ® WF PS*Lupasol ® WF 5.3 Yes 0 2.3 Lupasol ® SN PS* PAMAM-G6.0-NH₂ 1.4 Yes 8 2.4PAMAM-G6.0-NH₂ PS* PAMAM-G6.0-NH₂ 5.1 Yes N/T*** 2.5 Lupasol ® SN PS*G-35 [70 kDa] 7.6 Yes 0 2.6 G-35 [70 kDa] PS* G-35 [70 kDa] 3.9 Yes 12.7 Lupasol ® SN PS* Lupasol ® WF 5.5 Yes N/T*** 2.8 Lupasol ® SK PS*Lupasol ® WF 3.5 Yes 3 2.9 Lupasol ® SK PS* Lupasol ® WF 8.6 Yes 1 2.10Lupasol ® SN PS* Epomin P-1050 8.4 Yes N/T*** 2.11 Lupasol ® SN PS*Lupasol ® WF 5.1 Yes 0 3.1 Lupasol ® SN PS* PAMAM-G6.0-NH₂ ^(c) 0.6 Yes5 3.2 Lupasol ® SK and PAMAM- PAMAM-G6.0-NH₂ ^(c) 3.8 Yes 1 Lupasol ® WFG6.0-NH₂ ^(c) 3.3 Lupasol ® WF PAMAM- PAMAM-G6.0-NH₂ ^(c) 4.0 Yes 1G6.0-NH₂ ^(c) 3.4 Lupasol ® SN PS* PAMAM-G6.0-NH₂ ^(c) 0.9 Yes 14  3.5Lupasol ® SN PS* Lupasol WF^(c) 3.5 Yes 7 3.6 Lupasol ® SN PS*PAMAM-G8.0-NH₂ ^(c) 0.6 Yes 12  3.7 Lupasol ® SN PS* PPI G5^(c) 1.7 Yes15  Uncoated ** N/A ** N/A ** N/A ** N/A No 94  PVC Clotting ** N/A *PS** N/A ** N/A ***N/T 95  control ^(a)Mean out of 2 values ^(b)Yes meansblue/violet staining, No means no staining at all ^(c)Deposition ofpre-prepared heparin hyperbranched conjugate *PS = Polysaccharide ** N/A= Not applicable ***N/T = Not tested

The number of platelets present after the blood was exposed to thenon-thrombogenic surface coating was calculated as a percentage of thenumber of platelets present before the blood was exposed to thenon-thrombogenic surface coating and is presented graphically forvarious samples in FIG. 8.

As seen in the table above and in FIG. 8, there is virtually no plateletloss (platelet loss indicates thrombosis) seen for the heparincontaining coatings tested. The uncoated PVC tubing and the surface withan outer layer of a sulfated polysaccharides (“clotting control”) showsignificant thrombosis in this experiment.

Example 6.2 Staining of a Non-Thrombogenic Surface Using Toluidine Blue

Tubing from Example 2.2 was subjected to Toluidine blue stain solution(200 mg/L in water) by immersing in the solution for 2 minutes followedby extensive water rinse. A blue/violet color was observed on thesurface of the luminal surface of the tubing indicating the covalentattachment of end-point functionalized heparin.

Example 6.3 Staining of a Non-Thrombogenic Surface Using Toluidine Blue

Tubing from Example 3.2 was subjected to Toluidine blue stain solution(200 mg/L in water) by immersing in the solution for 2 minutes followedby extensive water rinse. A blue/violet color was observed on thesurface of the luminal surface of the tubing indicating the covalentattachment of end-point functionalized heparin in the PAMAM-heparinconjugate. The staining of the luminal surface of the PVC-tubing can beseen in FIG. 9.

Example 6.4 Staining of a Non-Thrombogenic Surface Using Toluidine Blue

Tubing from Example 3.3 was subjected to Toluidine blue stain solution(200 mg/L in water) by immersing in the solution for 2 minutes followedby extensive water rinse. A blue/violet color was observed on thesurface of the luminal surface of the tubing indicating the covalentattachment of end-point functionalized heparin in the PAMAM-heparinconjugate.

Example 7 Preparation of Intermediates Example 7.1 Synthesis ofNHS-Activated 5-Hexenoic Acid

Hexenoic acid (1.00 g, 8.76 mmol) and hydroxysuccinimide (1.01 g, 8.76mmol) was dissolved in 10 mL of DCM and stirred at 0° C. A solution ofDCC (1.81 g, 8.76 mmol) in DCM (3 mL) was slowly added dropwise to thereaction mixture at 0° C. The reaction was left to stir over night whereafter the byproducts were filtered off and the remaining solution wasconcentrated using a rotor evaporator and dried under vacuum in oven.High purity of the obtained material was confirmed by ¹H and ¹³C NMR.

Example 8 Preparation of a Hydrophilic and Lubricious Coatings Example8.1 A hydrophilic and Lubricious Coating Comprising Lupasol® SK andLupasol® WF

QCM crystals were coated according to Example 1.7 using Lupasol® SK toobtain a 3 bilayer coating consisting of alternatively layers ofLupasol® SK and a sulfated polysaccharide. A layer of Lupasol® WF wassubsequently adsorbed to the sulfated polysaccharide in order to obtaina coating with a cationic hyperbranched polymer as the outermost layer.A 2 min water rinse was conducted in between each adsorption step. Thesecoatings were analyzed using contact angle (CA) measurements. A staticCA of 53.0° (mean out of two samples) revealed that a hydrophilic andlubricious coating was obtained.

Example 8.2 A hydrophilic and Lubricious Containing Coating ComprisingLupasol® SK, Lupasol® WF and Heparin

QCM crystals were coated according to Example 1.7 using Lupasol® SK toobtain a 3 bilayer coating consisting of alternatively layers ofLupasol® SK and a sulfated polysaccharide. A layer of Lupasol® WF wassubsequently adsorbed to the sulfated polysaccharide followed by a 1hour coupling step of nitrous degraded heparin (325 mg/L), from Example4.1, using a reducing agent (sodium cyanoborohydride, 2.5 wt % inwater). A 2 min water rinse was conducted in between each adsorptionstep. The fabricated lubricious coating was treated with aborate/phosphate solution to remove any potential ionically boundheparin prior to evaluation using contact angle (CA) measurements. Astatic CA of 23.5° (mean out of two samples) reveled that a hydrophilicand lubricious coating was obtained.

Example 9 Preparation of Drug Eluting Coatings Example 9.1 Incorporationof Doxorubicin Into a Heparinized Coating

Doxorubicin was incorporated into a coating on a QCM crystal, preparedessentially as Example 2.3, by placing the QCM crystal in a watersolution of doxorubicin (1 mg/25 mL of water). The loading step wasfollowed by careful rinsing of the drug loaded coating using water priorto fluorescent evaluation of the coating. The crystal was dried in avacuum oven prior to fluorescent evaluation. A strong red fluorescencecould be detected indicating that doxorubicin was successfullyincorporated into the coating.

Example 9.2 Incorporation of Doxorubicin and the Subsequent Release Froma Coating Comprising Heparinized PAMAM-G6.0-NH₂ Dendrimer, Lupasol® SKand Lupasol® WF

Doxorubicin was incorporated into a coating on a QCM crystal, preparedessentially as Example 3.2, by placing the QCM crystal in a watersolution of doxorubicin (1 mg/25 mL of water). The loading step wasfollowed by careful rinsing of the drug loaded coating using water priorto fluorescent evaluation of the coating. The crystal was dried in avacuum oven prior to fluorescent evaluation. A strong red fluorescencecould be detected indicating that doxorubicin was successfullyincorporated into the coating. The drug loaded coating was subjected toa 2M NaCl-solution and a final water rinse followed by drying in vacuumoven prior to an additional fluorescent microscopy evaluation. The lackof red fluorescence indicates that the doxorubicin had eluted out fromthe coating.

Example 9.3 Incorporation of Doxorubicin and the Subsequent Release Froma Coating Comprising Heparinized PAMAM-G6.0-NH₂ Dendrimer and Lupasol®WF

Doxorubicin may be incorporated into a QCM crystal, prepared essentiallyas Example 3.3, by placing the QCM crystal in a water solution ofdoxorubicin (1 mg/25 mL of water) followed by careful rinsing of thedrug loaded coating using water prior to fluorescent evaluation of thecoating. A strong red fluorescence indicates that doxorubicin wassuccessfully incorporated into the coating. The drug loaded coating wassubjected to a 2M NaCl-solution followed by drying in vacuum oven priorto an additional fluorescent microscopy evaluation. The lack of redfluorescence indicated that the doxorubicin had been eluted out from thecoating.

Example 10 Biocompatibility Study Preparation of a Biocompatible Surfaceon a HDPE (High Density Poly Ethylene)

HDPE sheets (30 cm², USP reference standard) were cleaned withisopropanol and an oxidising method. The sheets were then primed as inExample 1 with 3 bilayers ending with sulfated polysaccharide. Thepriming layers were reacted as in Example 2 with a hyperbranchedpolyamine followed by a coupling step where functionalized heparin wasattached or as in Example 3 first with a polyamine layer followed by aheparin functionalized hyperbranched polymer with net negative charge.The coating was performed by immersing the materials into the coatingsolutions. The coatings were found to be non-toxic in a cytotoxicitytesting using the Minimal Essential Medium (MEM) elution test asdescribed in IS010993 (see Example 10.1).

These results demonstrate the non-toxic biocompatible properties of theevaluated surface.

Example 10.1 Table of Biocompatibility

Neg. Hyperbranched Polyamine charged polymer in Example in polymer inouter coating Not No. underlayer underlayer layer Passed passed 2.2Lupasol ® PS* Lupasol ® WF Yes WF 3.6 Lupasol ® PS* PAMAM-G8.0- Yes SNNH₂ ^(a) 3.7 Lupasol ® PS* PPI G5^(a) Yes SN *PS = Polysaccharide^(a)Deposition of pre-prepared heparin hyperbranched conjugate

Example 11 Hemo-Compatibility of EO Sterilized Coatings ComprisingHyperbranched Polymers EO Sterilization

Differently coated substrates with a heparin functionalizedhyperbranched polymer in the outer coating layer prepared as describedin Examples 2 or 3 were subjected to sterilization by exposure toethylene oxide (EO). The EO-sterilization was performed using a standardsterilization process used for medical devices.

Blood Loop Evaluation Test (For Measurement of Platelet Loss)

The EO-sterilized and washed tubings were incubated in a Chandler loopmodel performed essentially according to Andersson et al. (Andersson,J.; Sanchez, J.; Ekdahl, K. N.; Elgue, G.; Nilsson, B.; Larsson, R. JBiomed Mater Res A 2003, 67(2), 458-466), see Example 6. As seen in thetable below there is virtually no platelet loss (platelet loss indicatesthrombosis) seen for the EO sterilized heparin coatings prepared usingthe hyperbranched heparin conjugates prepared according to example 2 and3. The uncoated PVC tubing and the clotting control (surface with anouter layer of sulfated polysaccharides not binding antithrombin) showsignificant thrombosis in this experiment.

Example 11.1 Presentation of Coating Stability in Terms of BloodPlatelet Loss After EO Sterilization

Neg. Hyperbranched Platelets loss Platelets Polyamine charged polymer in[%] loss [%] in polymer in outer coating Pre EO- Post EO- Example No.underlayer underlayer layer sterilization sterilization 2.1 Lupasol ®PS* Lupasor ® WF 0 6 SN 2.7 Lupasol ® PS* Lupasol ® WF N/T** 8 SN 3.4Lupasol ® PS* PAMAM-G6.0- 14 6 SN NH₂ ^(a) 3.5 Lupasol ® PS* LupasolWF^(a) 7 0 SN 3.6 Lupasol ® PS* PAMAM-G8.0- 12 8 SN NH₂ ^(a) 3.7Lupasol ® PS* PPI G5^(a) 15 7 SN Uncoated N/A*** N/A*** N/A*** 97 N/T**PVC Clotting N/A*** N/A*** N/A*** 96 N/T** control *PS = Polysaccharide**N/T = Not tested ***N/A = Not applicable ^(a)Deposition ofpre-prepared heparin hyperbranched conjugate

These results demonstrate that the non-thrombogenic properties of thestable surfaces prepared according to the invention are retained inspite of exposure to rigorous sterilization conditions.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

All patents and patent applications mentioned throughout thespecification of the present invention are herein incorporated in theirentirety by reference.

The invention embraces all combinations of preferred and more preferredgroups and suitable and more suitable groups and embodiments of groupsrecited above.

The invention claimed is:
 1. A device having a surface comprising alayered coating wherein the outer coating layer comprises a plurality ofcationic hyperbranched polymer molecules characterized by having (i) acore moiety of molecular weight 14-1,000 Da (ii) a total molecularweight of 1,500 to 1,000,000 Da (iii) a ratio of total molecular weightto core moiety molecular weight of at least 80:1 and (iv) functional endgroups, whereby one or more of said functional end groups have ananti-coagulant entity covalently attached thereto, wherein thehyperbranched polymer is a polyamidoamine (PAMAM)dendrimer, wherein thelayered coating comprises one or more coating bilayers of cationicpolymer and anionic polymer, the innermost layer being a layer ofcationic polymer, wherein one or more of the layers of the layeredcoating other than the outer coating layer comprises a cationichyperbranched polymer molecule which is a PAMAM dendrimer characterizedby having (i) a core moiety of molecular weight 14-1,000 Da (ii) a totalmolecular weight of 1,500 to 1,000,000 Da (iii) a ratio of totalmolecular weight to core moiety molecular weight of at least 80:1 and(iv) functional end groups which are optionally derivatised.
 2. A deviceaccording to claim 1 wherein the anti-coagulant entities are heparinmoieties.
 3. A device according to claim 2 wherein the heparin moietiesare single point attached to the hyperbranched polymer molecules.
 4. Adevice according to claim 2, wherein the heparin moieties are attachedto the hyperbranched polymer molecules via the reducing end of theheparin moiety.
 5. A device according to claim 1, wherein thehyperbranched polymer has a molecular weight of 25,000 to 200,000 Da. 6.A device according to claim 1, wherein the hyperbranched polymermolecules are cross-linked to form aggregates of two or morehyperbranched polymer molecules.
 7. A device according to claim 1 whichis a medical device.
 8. A device according to claim 1 wherein thelayered coating of the device comprises one or more beneficial agentsbesides the anti-coagulant entities.
 9. A process for the manufacture ofa device according to claim 1, the process comprising, in any order: i.reacting a plurality of functional end groups of hyperbranched polymermolecules with anti-coagulant entities such that each hyperbranchedpolymer molecule is covalently linked to a plurality of anti-coagulantentities; and ii. attaching the hyperbranched polymer molecules to thesurface of a device.
 10. A non-thrombogenic device which is obtainableby a process comprising: (a) treating a device to present a surfacecoating comprising an outer coating layer comprising cationichyperbranched polymer molecules characterized by having (i) a coremoiety of molecular weight 14-1,000 Da (ii) a total molecular weight of1,500 to 1,000,000 Da and (iii) a ratio of total molecular weight tocore moiety molecular weight of at least 80:1 and bearing functional endgroups; (b) reacting at least one or more of said functional end groupswith molecules of an anti-coagulant entity which is functionalized tobear groups which are capable of reacting with the reactive functionalgroups on the hyperbranched cationic polymer; thereby to attach theanti-coagulant entity to the hyperbranched cationic polymer, wherein thehyperbranched polymer is a PAMAM dendrimer, wherein the coatingcomprises one or more coating bilayers of cationic polymer and anionicpolymer, the innermost layer being a layer of cationic polymer, whereinone or more of the layers of the layered coating other than the outercoating layer comprises a cationic hyperbranched polymer molecule whichis a PAMAM dendrimer characterized by having (i) a core moiety ofmolecular weight 14-1,000 Da (ii) a total molecular weight of 1,500 to1,000,000 Da (iii) a ratio of total molecular weight to core moietymolecular weight of at least 80:1 and (iv) functional end groups whichare optionally derivatised.
 11. A non-thrombogenic device which isobtainable by a process comprising: (a) treating a device to present apositively charged polymer surface layer; and (b) associating with saidpolymer surface layer functionalized cationic hyperbranched polymermolecules characterized by having (i) a core moiety of molecular weight14-1,000 Da (ii) a total molecular weight of 1,500 to 1,000,000 Da and(iii) a ratio of total molecular weight to core moiety molecular weightof at least 80:1 and bearing a multiplicity of negatively chargedanti-coagulant entities such as heparin moieties and wherein saidfunctionalized hyperbranched cationic polymer has a net negative chargeand is a PAMAM dendrimer, wherein the coating comprises one or morecoating bilayers of cationic polymer and anionic polymer, the innermostlayer being a layer of cationic polymer, wherein one or more of thelayers of the layered coating other than the outer coating layercomprises a cationic hyperbranched polymer molecule which is a PAMAMdendrimer characterized by having (i) a core moiety of molecular weight14-1,000 Da (ii) a total molecular weight of 1,500 to 1,000,000 Da (iii)a ratio of total molecular weight to core moiety molecular weight of atleast 80:1 and (iv) functional end groups which are optionallyderivatised; or (a) treating a device to present a negatively chargedpolymer surface layer; and (b) associating with said polymer surfacelayer functionalized cationic hyperbranched polymer moleculescharacterized by having (i) a core moiety of molecular weight 14-1,000Da (ii) a total molecular weight of 1,500 to 1,000,000 Da and (iii) aratio of total molecular weight to core moiety molecular weight of atleast 80:1 and bearing one or more negatively charged anti-coagulantentities such as heparin moieties and wherein said functionalizedhyperbranched polymer has a net positive charge and is a PAMAMdendrimer, wherein the coating comprises one or more coating bilayers ofcationic polymer and anionic polymer, the innermost layer being a layerof cationic polymer, wherein one or more of the layers of the layeredcoating other than the outer coating layer comprises a cationichyperbranched polymer molecule which is a PAMAM dendrimer characterizedby having (i) a core moiety of molecular weight 14-1,000 Da (ii) a totalmolecular weight of 1,500 to 1,000,000 Da (iii) a ratio of totalmolecular weight to core moiety molecular weight of at least 80:1 and(iv) functional end groups which are optionally derivatised.
 12. Adevice according to claim 1, wherein the ratio of total molecular weightto core moiety molecular weight is at least 100:1.